Isolation of neurotrophins from a mixture containing other proteins and neurotrophin variants using hydrophobic interaction chromatography

ABSTRACT

Methods are provided for large scale purification of neurotrophins, including mature NGF, suitable for clinical use. The methods provide means to separate neurotrophins from various less desirable misprocessed, misfolded, size, glycosylated, or charge forms. Compositions of neurotrophins, including mature NGF, substantially free of these variants are also provided.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 09/675,503, filed on Sep.29, 2000, now U.S. Pat. No. 6,423,831, which is a continuation ofapplication Ser. No. 09/363,573, filed on Jul. 29, 1999, now U.S. Pat.No. 6,184,360, which is a continuation of application Ser. No.08/970,865 filed on Nov. 14, 1997, now U.S. Pat. No. 6,005,081 whichclaims priority under USC Section 119(e) to Provisional Application Ser.No. 60/030,838 filed on Nov. 15, 1996 and Provisional Application Ser.No. 60/047,855 filed May 29, 1997, all of which are incorporated hereinby reference.

FIELD OF THE INVENTION

This invention relates to an improved method for purifyingneurotrophins, particularly those in the NGF-family, more particularlynerve growth factor (NGF) and neurotrophin-4/5 (NT-4/5), andneurotrophin-3 (NT-3) from variants, impurities, and contaminantsassociated therewith, particularly when produced by bacterial ormammalian cell fermentation.

BACKGROUND OF THE INVENTION

The production of large quantities of relatively pure, biologicallyactive polypeptides and proteins is important economically for themanufacture of human and animal pharmaceutical formulations, enzymes,and other specialty chemicals. For production of many proteins,recombinant DNA techniques have become the method of choice becauselarge quantities of exogenous proteins can be expressed in mammalianhost cells and, bacteria, and other host cells.

The primary structure of a mammalian NGF (mouse NGF) was firstelucidated by Angeletti and Bradshaw, Proc. Natil. Acad. Sci. USA68:2417 (1971). The primary structure of its precursor, pre-pro-NGF, hasbeen deduced from the nucleotide sequence of the mouse NGF cDNA (Scottet al. Nature 302:538 (1983); Ullrich et al. Nature 303:821 (1983)).

The homologous human NGF (hNGF) gene has also been identified (Ullrich,Symp. on Quan. Biol., Cold Spring Harbor 48:435 (1983); U.S. Pat. No.5,288,622, issued Feb. 22, 1994, which is incorporated herein byreference). Its homology to the mouse NGF is about 90% and 87%, on theamino acid and nucleotide sequence levels, respectively. Due to thescarcity of naturally-occurring human NGF, it has not been prepared fromnatural sources in quantities sufficient to biochemically characterizein fine detail.

Additional neurotrophic factors related to NGF have since beenidentified. These include brain-derived neurotrophic factor (BDNF)(Leibrock, et al., Nature, 341:149-152 (1989)), neurotrophin-3 (NT-3)(Kaisho, et al., FEBS Lett., 266:187 (1990); Maisonpierre, et al.,Science, 247:1446 (1990); Rosenthal, et al., Neuron, 4:767 (1990)), andneurotrophin 4/5 (NT-4/5) (Berkmeier, et al., Neuron, 7:857-866 (1991)).GDNF, a distant member of the TGF-.beta. super family, and neurturin(“NTN”) are two, recently identified, structurally related, potentsurvival factors for sympathetic sensory and central nervous systemneurons (Lin et al. Science 260:1130-1132 (1993); Henderson et al.Science 266:1062-1064 (1994); Buj-Bello et al., Neuron 15:821-828(1995); Kotzbauer et al. Nature 384:467-470 (1996)).

Producing recombinant protein involves transfecting host cells with DNAencoding the protein and growing the cells under conditions favoringexpression of the recombinant protein. The prokaryote E. coli is hasbeen a favored host because it can be made to produce recombinantproteins in high yields at low cost. Numerous U.S. patents on generalbacterial expression of DNA encoding proteins exist, including U.S. Pat.No. 4,565,785 on a recombinant DNA molecule comprising a bacterial genefor an extracellular or periplasmic carrier protein and non-bacterialgene; U.S. Pat. No. 4,673,641 on co-production of a foreign polypeptidewith an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on anexpression vector with a trp promoter/operator and trp LE fusion with apolypeptide such as IGF-I; U.S. Pat. No. 4,795,706 on expression controlsequences to include with a foreign protein; and U.S. Pat. No. 4,710,473on specific circular DNA plasmids such as those encoding IGF-I.

Genetically engineered bio-pharmaceuticals are typically purified from asupernatant containing a variety of diverse host cell contaminants. NGF,in particular, has been reportedly purified to varying extent withvarying degrees of effort and success using a number of differentmethods. See for example, Longo et al., IBRO Handbook, vol. 12, pp 3-30(1989); U.S. Pat. No. 5,082,774, which discloses CHO cell production ofNGF; Bruce and Heinrich (Neurobio. Aging 10:89-94 (1989)); Schmelzer etal. J Neurochem. 59:1675-1683(1992); Burton et al., J Neurochem.59:1937-1945(1992)). These efforts have been primarily at laboratoryscale.

However, preparative isolation of recombinant human NGF resulting inpharmaceutical purity and high yield, essentially free of variants, haseluded the art.

Accordingly, there is a need in the art for an efficient protocol forselectively separating neurotrophins, particularly NGF and NGF-family ofneurotrophins, from their variants and other molecules, and from otherpolypeptides with high pI. The process of purifying neurotrophins atlarge scale should be applicable to starting material from varyingsources, including fermentation broth, lysed bacterial or mammaliancells, to supply clinical needs. Furthermore, as the present inventorshave discovered previously unknown, difficult-to-separate neurotrophinvariants, for example NGF variants, the methods presented herein areparticularly useful to provide commercially useful amounts ofrecombinant neurotrophins, including human NGF (rhNGF), rhNT-3, andrhNT-4/5 and desirable genetically engineered mutants thereof, that aresubstantially free of undesirable variants. These and other objects ofthe invention will now be apparent to one of ordinary skill in the art.

SUMMARY OF THE INVENTION

In one embodiment of the invention a process for purifying aneurotrophin, particularly one in the NGF-family, including NGF, NT-3,NT-4/5, and BDNF which share recognition by a highly homologous familyof receptors (trks), preferably rhNGF, rNT-3, rhNT-4/5, rhBDNF ordesirable genetically engineered forms thereof, by the use ofhydrophobic interaction chromatography (HIC) is provided. In view of thediscovery by the present inventors of certain undesirable neurotrophinvariants arising from recombinant production of a neurotrophin, asreported herein, the use of HIC can separate chemically different oreven misfolded forms of a neurotrophin from the desired correctlyfolded, intact neurotrophin. Variants that can be removed are those thatdiffer from the mature, correctly folded neurotrophin in hydrophobicity,including partially processed precursor sequences, glycosylated matureand precursor-containing forms (when present from eukaryotic cellculture), and misfolded and partially folded variants (generally frombacterial cell culture when in vitro folding steps are used). Forexample, HIC is particularly useful to remove partially processedprecursor sequences of NGF, glycosylated species of NGF and precursor(when present from eukaryotic cell culture), and misfolded and partiallyfolded variants (generally from bacterial cell culture and in vitrofolding steps) from mixtures of mature NGF. NGF has one N-linkedglycosylation site at Asn45. In the case of bacteria-expressed, refoldedrhNT-4/5, HIC separates correctly folded NT-4/5 from incorrectly foldedforms. As a result of the process described herein the neurotrophin isessentially free of these variants. For neurotrophin purification,preferably the HIC resin functional group is a phenyl group, while octyland butyl groups can be useful. Particularly preferred embodimentsinclude HIC resins Phenyl Toyopearl, Phenyl Sepharose Fast Flow LowSubstitution, TSK-Phenyl 5PW, or the like.

In another embodiment is provided a process for purifying aneurotrophin, particularly one in the NGF-family, preferably rhNGF,rNT-3, rhNT-4/5 or desirable genetically engineered forms thereof, bythe use of preparative cation-exchange chromatography, which separatescharge-modified variants, such as oxidized, isoasp and deamidated formsfrom mature neurotrophin. Particularly preferred embodiments useSP-Sepharose High Performance, Fractogel EMD SO3, or polyaspartic acidresin, of which PolyCAT A is particularly preferred. Most preferably atlarge scale SP-Sepharose High Performance or Fractogel EMD SO3 resinsare used.

In yet another embodiment of the invention both HIC and cation-exchangechromatography are used to prepare a composition of a desiredneurotrophin, for example recombinant mature NGF, preferably human NGF,that is substantially homogenous, i.e., substantially free of bothprocess and charge variants, e.g. misfolded and chemical variants, andis also substantially pure with regard to protein content.

In one embodiment of this invention an improved process for separatingneurotrophins, particularly those of the NGF-family, preferablyrecombinant human NGF, NT-3, NT-4/5, and their desirable geneticallyengineered forms, from related undesirable variants, e.g. fermentation,protease-cleaved variants, glycosylation variants, misfolded variants,by means of reversed-phase liquid chromatography is provided. Morepreferably the NGF is the 120/120 or 118/118 homodimer form. As a resultof the process described herein the neurotrophin is most preferablyessentially free of variants.

In another embodiment a process for purifying neurotrophins,particularly those of the NGF-family, from related variants usingelution conditions involving physiological pH is provided.

In still another embodiment a process for purifying a neurotrophin thatresults in considerable improvement in its homogeneity is provided.

In another embodiment, the invention provides a process for separatingNGF-family neurotrophins from variants thereof comprising:

a) loading a buffer containing the neurotrophin and its variant at a pHof about 5 to 8 onto a hydrophobic interaction chromatography column;

b) washing the column

c) eluting the neurotrophin with a buffer at a pH of about 5 to 8;

d) loading the neurotrophin-containing eluant onto a cation-exchangechromatography column at a pH from about 5 to 8; and

e) eluting the neurotrophin from the column with a buffer at a saltgradient at a pH at about 5 to 8, preferably pH 6. The neurotrophin ismost preferably rhNGF.

In one embodiment of the invention is provided a silica gelchromatography step that efficiently removes host cell proteins from theneurotrophin fraction, which is preferably an NGF fraction.

In one embodiment of the invention, a process step is provided in which120 amino acid NGF is subjected to trypsin-like protease treatment toselectively remove the terminal RA dipeptide from the VRRA C-terminal,to yield the 118 species. An immobilized trypsin column is preferred.

The invention also relates to the neurotrophin composition andformulation prepared by the processes of the invention and to uses forthe composition and formulation. Provided is a composition ofneurotrophin that is substantially homogenous, i.e., substantially freeof both process and charge variants, e.g. misfolded and chemicalvariants, and is also substantially pure with regard to protein content.Preferably, mature human NGF, mature human or rat NT-3, and mature humanNT-4/5 are provided in this form. In a preferred embodiment the NGF isthe 120 species, and more preferably the 118 form, most preferably as ahomodimer, e.g., 118/118.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an SP-Sepharose HP chromatogram. An NGF-containingmixture from a 12 kL fermentation, after HIC chromatography, was loadedonto a SP-Sepharose High Performance resin (column dimension 1.0×35 cm;a 27.5 ml bed volume) of 25 omnifit SPSHP resin. Buffer A was 0.2 MNaCl, 20 mM succinate, pH 6.0 (23 ms). Buffer B was 0.7M NaCl, 20 mMSuccinate, pH 6.0 (63 ms). The column was first equilibrated in BufferA. The HIC pool of 345 ml at 0.24 mg/ml was adjusted to 25 mM succinate,pH 6.0 (17 ms) of which 362 ml was loaded giving a 3 mg/ml resin load;82.5 mg NGF was loaded. Load rate was 40 cm per hour. NGF was elutedusing a 22 column volume gradient from 30% to 80% Buffer B (0.35 to 0.60M NaCl; 37 to 57 ms). Elution rate was 60 cm per hour. Absorbance units(280 nm) and mS/cm were plotted versus fraction numbers and elutionvolume in ml. Fractions containing NGF were determined and pooled. Inthis case fractions 43 to 60 were pooled to obtain 99 ml sample at 0.38mg/ml NGF, giving about a 46% recovery.

FIG. 2 depicts an SP-NPR HPLC cation-exchange (HPIEX) analysis of thePhenyl Sepharose Fast Flow pool and the SP-Sepharose HP pool.Chromatogram for each are marked.

FIG. 3 depicts an C4 RP-HPLC analysis of selected fractions (main pool,leading edge and trailing edge of main peak pool) from the SP-SepharoseHP chromatography step. The three signals are overlaid and are asmarked. As can be seen, the main peak (containing mature NGF) isseparated from several lesser peaks that contain variant NGFs.

FIG. 4 depicts the sequence of human prepro-NGF (SEQ ID NO: 1).Indicated on the figure is the first amino acid of mature NGF(position 1) and the last amino acid of the 120 NGF form (position 120).A preferred NGF amino acid sequence is that of the mature 118 form (fromposition 1 to position 118). Also shown are variant forms: mature 120(position 1 to position 120); mature 117 (position 1 to 117); R120(position −1 to 120); and sites for other misprocessing that occurs atthe N- and C-terminal ends, including mature 114 (amino acids 1 to 114),115 (amino acids 1 to 115) and 117 (amino acids 1 to 117) variants. Amajor misprocessed variant, proteolytically misprocessed, has N-terminalcleavage between amino acids R(−39) and S(−38) in the pro sequence ofNGF. The likely initiation Met are underlined. Other N-terminal variantsinclude truncated forms of NGF, with the most common having cleavageoccurring between amino acids H8 and R9 and between R9 and G10.

FIG. 5 depicts the amino acid sequences of neurotrophins human NGF (SEQID NO: 2), mouse NGF (SEQ ID NO: 3), BDNF (SEQ ID NO: 4), NT-3 (SEQ IDNO: 5) and NT-4/5 (SEQ ID NO: 6). The 15 boxed regions indicate thehomologous cysteine-containing regions involved in the cysteine knotmotif (De Young et al., Protein Sci. 5 (8): 1554-66 (1996)).

FIG. 6 depicts the chromatography pattern of rhNT-4/5 on DEAE-SepharoseFast Flow (DEFF) resin column. The chromatogram marked “NT45DE1:1_UV” isa UV absorbance measurement at 280 nm. The chromatogram marked“NT45DE1:1_Cond1” is a conductivity measurement of eluting fractions.The neurotrophin-containing fractions that were pooled are indicated bythe horizontal arrow marked “Pool.”

FIG. 7 depicts the chromatography pattern of rhNT-4/5 on SP-SepharoseFast Flow resin column. The chromatogram marked “NT45SFF1:1_UV” is a UVabsorbance measurement at 280 nm. The chromatogram marked“NT45SFF1:1_Cond1” is a conductivity measurement of eluting fractions.The neurotrophin-containing fractions that were pooled are indicated bythe horizontal arrow marked “Pool.”

FIG. 8 depicts a typical preparative C4-RP-HPLC chromatography patternof rhNT-4/5 under conditions described in the text. Absorbance at 280 nmwas monitored. The neurotrophin-containing fractions that were pooledare marked with a horizontal arrow marked “Pool.”

FIG. 9 provides the analytical HPLC chromatography pattern of NT4/5samples monitored during refolding at the times indicated. Columnconditions are described in the text. “NT-4/5 Std.” indicates theelution pattern of a correctly folded, intact NT-4/5 used as a standard.The pattern marked “SSFF (0.5 m)” depicts the analysis of NT-4/5 elutedwith 0.5M NaCl from the S-Sepharose Fast Flow column prior to refolding.As NT-4/5 refolds, the elution pattern approaches that of the standard.

FIG. 10 depicts a chromatogram showing separation of intact, correctlyfolded NT-4/5 from misfolded variants on a hydrophobic interactionchromatography column, Phenyl Toyopearl 650M column. Misfolded variantsthat are less hydrophobic than correctly folded NT-4/5 elute in theflow-through, while misfolded variants (Peak B) that are morehydrophobic elute at an organic solvent concentration higher than thatneeded to elute correctly folded NT-4/5 (Peak A).

FIG. 11 depicts the elution pattern of NT-4/5 and variants from a cationexchange resin, SP-Sepharose. Absorbance at 280 nm was monitored. Peak Acontains carbamylated and clipped variants, hile Peak B contains intact,correctly folded NT-4/5. NT-4/5-containing fractions that were pooledare indicted by the horizontal arrow marked “Pool.”

FIG. 12 depicts a typical preparative poly CAT A resin chromatographypattern of rhNT-4/5 under conditions described in the text.

FIG. 13 depicts a 16% SDS-PAGE (Tris-glycine system, pre-poured, fromNovex, Inc., San Diego, Calif.) analysis under reducing conditions toassess purity and homogeneity of samples taken from the indicated stepsof the rhNT4/5 purification process described in the text. The gel wasstained with Coomassie-R250 to detect protein (Andrews, Electrophoresis,Oxford University Press: New York, 1986). The lane marked “DE Load”contains a sample of the PEI-mixture that was loaded onto theDE-Sepharose Fast Flow column; lane marked “DE FT” contains a sample ofthe flow through from the DE-Sepharose Fast Flow column; lane “S Pool”contains a sample from the pooled fractions containing NT-4/5 elutedfrom the S-Sepharose Fast Flow resin column, prior to refolding; lane“Refold Pool” contains a sample of the pool after refolding wascompleted; lane “C4 Pool” contains a sample of the pooled fractionsafter preparative C4 RP-HPLC; and lane “PolyCAT A Pool” contains asample from the pooled NT-4/5-fractions from the PolyCAT A HPLC column.

FIG. 14 depicts the UV absorbance pattern of fractions from S-SepharoseFast Flow chromatography of a mixture containing bacterially-produced,sulfonylated rhNT-3.

FIG. 15 depicts Macroprep High S cation-exchange chromatography of amixture containing in vitro re-folded forms of rhNT-3. The resin waspurchased from Biorad. Column dimensions were 9×9 cm. A 700 ml SSFF poolcontaining refolded (after 36 hour refold), pH 6.8, was loaded onto theMacroprep column at a flow of about 310 ml/min. Conditions are given inthe text.

FIG. 16 depicts a Phenyl Sepharose Fast Flow High Substitution(hydrophobic interaction chromatography) chromatography ofcation-exchange-purified, refolded rhNT-3 to remove misfolded variants.

FIG. 17 depicts a SP-Sepharose High Performance chromatography of theHIC-rhNT-3 pool.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

As used herein, “neurotrophin” refers to a neurotrophin, preferably anNGF-family neurotrophin, including NGF, NT-3, NT-4/5, and BDNF, from anyspecies, including murine, bovine, ovine, porcine, equine, avian, andpreferably human, in native sequence or in a genetically engineeredform, and from any source, whether natural, synthetic, or recombinantlyproduced. For example, “NGF” refers to nerve growth factor from anyspecies, including murine, bovine, ovine, porcine, equine, avian, andpreferably human, in native sequence or in genetically engineeredvariant form, and from any source, whether natural, synthetic, orrecombinantly produced. Preferably, the neurotrophin is recombinantlyproduced. In a preferred method, the neurotrophin is cloned and its DNAexpressed, e.g., in mammalian cells, in bacterial cells. The processesand methods taught herein can also be applied to the neurotrophins GDNFand neurturin.

Preferred for human use is human native-sequence, mature NGF, morepreferably a 120 amino acid sequence, and even more preferably a 118amino acid sequence. More preferably, this native-sequence NGF isrecombinantly produced. The preferred amino acid sequence for humanpre-pro-NGF and human mature NGF are provided by U.S. Pat. No.5,288,622, which is specifically incorporated herein by reference. The120 amino acid form, without additional post-translationalmodifications, is a preferred form in the homodimer form (i.e.,120/120). Even more preferred is the 118 form, without additionalpost-translational modifications, particularly as a homodimer (i.e.,118/118).

By “substantially pure” is meant a degree of purity of totalneurotrophin, e.g., NGF, to total protein where there is at least 70%neurotrophin, more preferably at least 80%, and even more preferablyincreasing to at least 90%, 95% or 99%. A particularly preferred purityis at least 95%. By “essentially pure” is meant that the composition isat least 90% or more pure for the desired neurotrophin.

By “substantially free of neurotrophin variant” is meant a compositionin which the percent of desired neurotrophin species to totalneurotrophin (including less desirable neurotrophin species) is at least70% desired neurotrophin species, more preferably at least 80%, and evenmore preferably increasing to at least 90%, 93%, 95% or 99%. By“essentially free” is meant that the composition contains at least 90%or more desired neurotrophin. A particularly preferred level is at least95% desired neurotrophin, e.g., correctly folded, intact 118/118 rhNGF,species. The other undesirable species or forms may be misprocessedforms or chemical variants, e.g. altered charge variants, resulting fromthe fermentation or purification process, or preferably all of theforegoing, as disclosed herein. For example, when NGF is folded in vitroafter synthesis in bacteria, “species” or “variants” can includemisfolded or partially folded forms.

By “misfolded” variant is meant a variant of the neurotrophin whichdiffers from the neurotrophin by the pairing of its cysteine residues orby the particular cysteine residues which are free or blocked. Misfoldedvariants can also have the same cysteine pairing as the neurotrophin buthave a different three dimensional conformation resulting frommisfolding.

By “chemical” variant is meant a variant that differs chemically fromthe neurotrophin, for example by having an altered charge, bycarbamylation, deamidation, oxidation, glycosylation, or proteolyticcleavage.

Buffers for the column aspect of this invention generally have a pH inthe range of about 5 to 8. Buffers that will control the pH within thisrange include, for example, citrate, succinate, phosphate, MES, ADA,BIS-TRIS Propane, PIPES, ACES, imidazole, diethylmalonic acid, MOPS,MOPSO, TES, TRIS buffer such as TRIS-HCl, HEPES, HEPPS, TRICINE, glycineamide, BICINE, glycylglycine, and borate buffers. A preferred buffer isa MOPSO buffer.

As used herein, “alcohols” and “alcoholic solvents” are meant in thesense of the commonly used terminology for alcohol, preferably alcoholswith 1 to 10 carbon atoms, more preferably methanol, ethanol,iso-propanol, n-propanol, or t-butanol, as well as glycerol, propyleneglycol, ethylene glycol, hexylene glycol, polypropylene glycol, andpolyethylene glycol, and most preferably ethanol or iso-propanol. Suchalcohols are solvents that, when added to aqueous solution, increase thehydrophobicity of the solution by decreasing solution polarity.

MOPSO is 3-(N-Morpholino)-2-hydroxypropanesulfonic acid. HEPES isN-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid. Reagent alcohol is95 parts by volume (Specially Denatured Alcohol Formula 3A and 5 partsby volume isopropyl alcohol). MES is 2-(N-Morpholino)ethanesulfonicacid. UF/DF means ultrafiltration/diafiltration. TMAC istetramethylammonium chloride. TEAC is tetraethylammonium chloride.NGF-120 means full-length of 120/120 nerve growth factor. NGF-118 meanshomodimeric mature NGF molecule of 118 residues. Oxidized NGF means NGFvariant molecule, Metsulfoxide.sub.37, which is reported herein to beabout 80% as biologically active as mature, native NGF. Isoasp NGF meansNGF isomerized variant molecule, Asp93. Deamidated NGF means NGF havingAsn45 converted to Asp45. RNGF means an NGF molecule with an extraArginine residue at its N-terminus. CHO means Chinese hamster ovarycells.

Resins described herein include MACROPREP HIGH S Cation-exchange(BIO-RAD Laboratories; strong cation exchange; SO₃ functional group;nominal particle size of 50 m; nominal pore size of 1000 A); Silica gel(underivatized); Phenyl Sepharose Fast Flow Low Substitution (Pharmacia;highly cross-linked 6% agarose; particle size of 45-165 microns);SP-Sepharose HP (Pharmacia; highly cross-linked 6% agarose; particlesize of 34 microns); Phenyl Toyopearl 650 M (TosoHaas; particle size of40-90 microns); and Fractogel EMD SO₃−650 S (EM Separations, a U.S.associate of E. Merck (Germany); particle size of 25-40 m).

Modes for Carrying out the Invention

Neurotrophins belong to a family of small, basic proteins which play acrucial role in the development and maintenance of the nervous system.The first identified and probably best understood member of this familyis nerve growth factor (NGF). See U.S. Pat. No. 5,169,762, issued Dec.8, 1992. Recently, sequentially related but distinct polypeptides withsimilar functions to NGF have been identified. For example,brain-derived neurotrophic factor (BDNF), also referred to asneurotrophin-2 (NT2), was cloned and sequenced by Leibrock etal.(Nature, 341: 49-152 [1989]). Several groups identified aneurotrophic factor originally called neuronal factor (NF), and nowreferred to as neurotrophin-3 (NT3). (Ernfors et al., Proc. NatL. Acad.Sci. USA, 87:5454-5458 [1990]; Hohn et al., Nature, 344:339 [1990];Maisonpierre et al., Science, 247:1446 [1990]; Rosenthal et al., Neuron,4:767 [1990]; Jones and Reichardt, Proc. Natl. Acad. Sci. USA,87:8060-8064 [1990]; Kaisho et al., FEBS Lett., 266:187 [1990]).Neurotrophin-4/5 (referred to as either NT4 or NT5) has been identified(Hallbook et al., Neuron, 6:845-858 [1991]; Berkmeier et al., Neuron,7:857-866 [1991]; Ip et al., Proc. Natl. Acad. Sci, USA, 89: 3060-3064[1992]). U.S. Pat. No. 5,364,769, issued Nov. 15, 1994, discloses humanNT-4/5 and processes for its recombinant expression and is incorporatedherein by reference. Also reported are chimeric and pantropicneurotrophins, such as that reported in U.S. Pat. No. 5,488,099, issuedJan. 30, 1996, in Urfer et al., EMBO J 13(24):5896-909 (1994), and in WO95/33829, published Dec. 1, 1995 (incorporated herein by reference) inwhich the neurotrophin has been modified to bind to more than onereceptor or contains a receptor binding activity not normally present toa significant degree in the native neurotrophin. Of particular interestare neurotrophins designated MNTS-1 and D15A NT3. Also of particularinterest are neurotrophins having an NGF amino acid backbone butmodified to bind receptors other than trkA, such as trkB or trkC.Preferred are those in which amino acid substitutions have been made inNGF with an amino acid from a corresponding position in NT-3 that isresponsible for binding the trk receptor for NT-3. Such NGF mutants haveNT-3-like receptor binding activity while retaining NGF pharmacokineticsand purification behavior (Urfer, et al, Biochemistry 36(16):4775-4781(1997)). These NGF mutants can also lack trkA binding activity (Shih etal, J Biol. Chem. 269 (44):27679-86 (1994). Such NGF mutants areparticularly preferred neurotrophins for use in the invention describedherein.

The isolation of a recombinant human neurotrophin, e.g., rhNGF, involvesseparation of the protein from a variety of diverse host cellcontaminants. Each step involves special buffers that enable sufficientseparation to take place. The final or penultimate processing step for aneurotrophin is complicated by the presence of several neurotrophinvariants that co-purify using conventional chromatographic media. When arefolding step is included in the recovery and purification process, thevariants include misfolded forms of the neurotrophin. Variants can alsoinclude those that differ chemically from the neurotrophin, such ascarbamylated, deamidated, deamidated or proteolytically cleaved forms.In the case of NGF, these species consist primarily of dimericforms—homodimers, e.g., 120/120 or 117/117 when 118/118 is desired, orheterodimers, e.g., 120/118, 117/118—, chemically modifiedvariants—isoaspartate, mono-oxidized, glycosylation variants, N-terminaland C-terminal truncated forms, and dimers thereof.

The invention makes possible the large scale production ofneurotrophins, particularly rhNGF, in quantities sufficient fortherapeutic uses, such as, for example, treatment of Alzheimer'sdisease, peripheral neuropathies, including diabetic and AIDS-relatedneuropathies, and the like.

In view of the similarity in sequence and conformation between NGF andother neurotrophins, preferably those in the NGF-family, the methods ofthe present invention can be applied to prepare these neurotrophinssubstantially free of misprocessed, misfolded or partially folded,glycosylation, and/or charge variants. In the present invention, columnresins and conditions are identified that are favorable for selectivelyseparating neurotrophins from these and other closely related variants.Neurotrophins include NT-3, NT-4/5, NT-6, BDNF, and engineered forms,including heterodimeric, chimeric or pantropic forms thereof. Preferablythe neurotrophins are human or highly homologous to the human amino acidsequence, preferably greater than 80%, more preferably greater than 90%,and most preferably greater than 95% homologous to the human sequences.An engineered neurotrophin will retain at least 50% of the trk receptorbinding function of the native neurotrophin it mimics, preferably atleast 75%, and more preferably at least 80%. These engineered forms arethose that retain sufficient high-pI or hydrophobic character of thenative neurotrophin to retain a similar performance in the processesdescribed herein.

As described below, the processes described herein have beensuccessfully applied to rhNGF, rhNT-3 and rhNT-4/5. For example,rhNT-4/5, which was made in E. coli, was isolated in inclusion bodiesand reduced and solubilized from the inclusion bodies. The reducedNT-4/5 was partially purified by DE. Sepharose Fast Flow and byS-Sepharose Fast Flow chromatography. The S-Sepharose Fast Flow pool wasrefolded in a guanidine containing buffer for 24 hours. Misfolded formsof NT-4/5 were removed by chromatography as disclosed herein at largescale. The carbamylated and clipped (misprocessed forms) of NT-4/5 wereremoved by high performance cation-exchange chromatography by a PolyCatA HPLC resin or SP-Sepharose HP resin, in column format, at large scale.The purified rhNT-4/5 was ultrafiltered and diafiltered into an acidicbuffer for formulation.

One embodiment of the invention involves purifying a neurotrophin fromits related variants, usually after the neurotrophin has already beenpurified from most other impurities, typically at the final or nearfinal step before desalting or diafiltration prior to formulation. Therelated variants in the mixture can include not only variants residualfrom a fermentation, but also variants produced if the neurotrophin isdegraded on storage or during processing.

The neurotrophins suitable for use with embodiments of the invention canbe prepared by any means, but are preferably prepared recombinantly. Anucleic acid molecule coding for the neurotrophins discussed herein areavailable from several sources, for example, through chemical synthesisusing the known DNA sequence or by the use of standard cloningtechniques known to those skilled in the art. cDNA clones carrying theneurotrophin, e.g., hNGF coding sequence, can be identified by use ofoligonucleotide hybridization probes specifically designed based on theknown sequence of the neurotrophin.

Upon obtaining a molecule having the neurotrophin coding sequence, themolecule is inserted into a cloning vector appropriate for expression inthe chosen host cell. The cloning vector is constructed so as to providethe appropriate regulatory functions required for the efficienttranscription, translation and processing of the coding sequence.

If the neurotrophin is prepared recombinantly, suitable host cells forexpressing the DNA encoding the neurotrophin are prokaryote, yeast, orhigher eukaryotic cells. Suitable prokaryotes for this purpose includebacteria such as archaebacteria and eubacteria. Preferred bacteria areeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella; Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989); Pseudomonas such as P.aeruginosa; Streptomyces; Azotobacter; Rhizobia; Vitreoscilla; andParacoccus. Suitable E. coli hosts include E. coli W3110 (ATCC 27,325),E. coli 94 (ATCC 31,446), E. coli B, and E. coli X1776 (ATCC 31,537).These examples are illustrative rather than limiting.

Incorporated herein in its entirety is PCT publication WO 95/30686published Nov. 16, 1995. The publication is particularly relevant forits description of bacterial synthesis and in vitro folding of NGF. Theproducts from that process can be subjected to the purification methodsof the present invention.

Mutant cells of any of the above-mentioned bacteria may also beemployed. It is, of course, necessary to select the appropriate bacteriataking into consideration replicability of the replicon in the cells ofa bacterium. For example, E. coli, Serratia, or Salmonella species canbe suitably used as the host when well known plasmids such as pBR322,pBR325, pACYA177, or pKN410 are used to supply the replicon. E. colistrain W3110 is a preferred host or parent host because it is a commonhost strain for recombinant DNA product fermentation. Preferably, thehost cell secretes minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins endogenous to the host, with examples of such hostsincluding E. coli W3110 strain 1A2, which has the complete genotypetonAΔ; E. coli W3110 strain 9E4, which has the complete genotypetonAΔptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has thecomplete genotype tonA ptr3 phoAΔE15 Δ (argF-lac)169ΔdegPΔompT kan<r>;E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3phoA Δ E15 Δ (argF-lac)169 Δ degP Δ ompT Δ rbs7 ilvG kan r; E. coliW3110 strain 40 B4, which is strain 37D6 with a non-kanamycin resistantdegP deletion mutation; and an E. coli strain having mutant periplasmicprotease disclosed in U.S. Pat. No. 4,946,783 issued Aug. 7, 1990.

Human NGF has been expressed in E. coli. The isolation and sequence ofthe gene encoding the .beta.-subunit of hNGF and its expression as aheterologous protein in E. coli was described in U.S. Pat. No.5,288,622. The teachings therein are also suitable to provide mammaliancell produced mature human NGF. By using recombinant techniques, human.beta.-NGF was expressed free from other mammalian proteins. Expressionof the hNGF in E. coli using two genes which contain alteredamino-termini resulted in the expression of a fused protein, which wasdescribed by Iwai et al., Chem. Pharm. Bull. 34:4724 (1986).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable expression hosts for neurotrophin-encodingvectors. Saccharomyces cerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms. However, anumber of other genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe [Beach and Nurse,Nature, 290: 140 (1981); EP 139,383 published May 2, 1985];Kluyveromyces hosts [U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9: 968-975 (1991)] such as, e.g., K. lactis [MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 (1983)], K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum [ATCC 36,906; Van denBerg et al., Bio/Technology, 8: 135 (1990)], K. thermotolerans, and K.marxianus; yarrowia [EP 402,226]; Pichia pastoris [EP 183,070;Sreekrishna et al., J. Basic Microbiol., 28: 265-278 (1988)]; Candida;Trichodenna reesia [EP 244,234]; Neurospora crassa [Case et al., Proc.Natl. Acad. Sci. USA, 76: 5259-5263 (1979)]; Schwanniomyces such asSchwanniomyces occidentalis [EP 394,538 published Oct. 31, 1990]; andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium[WO 91/00357; published Jan. 10, 1991], and Aspergillus hosts such as A.nidulans [Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289(1983); Tilbum et al., Gene, 26: 205-221 (1983); Yelton et al.,Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984)] and A. niger [Kelly anHynes, EMBO J., 4: 475-479 (1985)].

Suitable host cells appropriate for the expression of the DNA encodingthe neurotrophin can also be derived from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture issuitable, whether from vertebrate or invertebrate culture. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. See, e.g., Luckow elal., Bio/Technology, 6:47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al., Nature, 315: 592-594 (1985). Avariety of viral strains for transfection are publicly available, e.g.,the L-1 variant of Autographa californica NPV and the Bm-5 strain ofBombyx mori NPV, and such viruses may be used herein, particularly fortransfection of Spodoptera frugiperda cells. Human NGF has been producedin insect cells as reported in U.S. Pat. No. 5,272,063, issued Dec. 21,1993.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which have been previously manipulated tocontain the DNA encoding the neurotrophin. During incubation of theplant cell culture with A. tumefaciens, the DNA encoding theneurotrophin is transferred to the plant cell host such that it istransfected, and will, under appropriate conditions, express the DNAencoding the neurotrophin. In addition, regulatory and signal sequencescompatible with plant cells are available, such as the nopaline synthasepromoter and polyadenylation signal sequences (Depicker et al, J. Mol.Appl. Gen., 1:561 (1982)). In addition, DNA segments isolated from theupstream region of the T-DNA 780 gene are capable of activating orincreasing transcription levels of plant-expressible genes inrecombinant DNA-containing plant tissue (EP 321,196 published Jun. 21,1989).

Examples of useful mammalian host cell lines are monkey kidney CVI linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line[293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36: 59 (1977)]; baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR [CHO, Urlaub and Chasin,Proc. Natl. Acad Sci. USA, 77: 4216 (1980)]; mouse sertoli cells [TM4,Mather, Biol. Reprod., 23: 243-251 (1980)]; monkey kidney cells (CV1,ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells[Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982)]; MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2). A preferred method isexpression in CHO cells. The exon of human NGF containing the prepro-NGFcan be used to achieve expression of secreted, mature NGF (including 118and 120 forms) using suitable promoters and vectors (U.S. Pat. No.5,288,622). Cultures of stable CHO cells stably transfected andsecreting mature forms of NGF are useful in the invention as discussedin the Examples herein.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. Transfection refers to the taking up of an expressionvector by a host cell whether or not any coding sequences are in factexpressed. Numerous methods of transfection are known to the ordinarilyskilled artisan, for example, CaPO4 and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., Molecular Cloning: A Laboratory Manual [New York: Cold SpringHarbor Laboratory Press, 1989], or electroporation is generally used forprokaryotes or other cells that contain substantial cell-wall barriers.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published Jun. 29, 1989. In addition, plants may betransformed using ultrasound treatment as described in WO 91/00358published Jan. 10, 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52: 456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued Aug. 16, 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al, J Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, etc.,may also be used. For various techniques for transforming mammaliancells, see Keown et al., Methods in Enzymology (1990) Vol. 185, pp.527-537, and Mansour et al., Nature, 336: 348-352 (1988).

Preferably, the gene for hNGF is inserted into the vector so as to haveavailable a methionine initiation codon, preferably one of the twomethionine initiation codons as identified by Ullrich et al. (Nature,303:821-825 (1983)). The hNGF gene (Ullrich, et.al., Cold Spring HarborSymposia on Quant. Biol. XLVIII, p. 435 (1983); U.S. Pat. No. 5,288,622)has two nearly adjacent methionines that are likely to be utilized astranslational initiation codons (position 1 refers to the N-terminalserine residue of mature hNGF). In contrast, the mouse submaxillarygland cDNA for NGF, the most thoroughly studied of the nerve growthfactors, has a methionine at position −187 in addition to those atpositions −121 and −119. In a preferred embodiment for expression inmammalian cells, the prepro-NGF sequence is present.

If prokaryotic cells are used to produce neurotrophin, they are culturedin suitable media in which the promoter can be constitutively orartificially induced as described generally, e.g., in Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, New York 1989). Any necessary supplements besides carbon,nitrogen, and inorganic phosphate sources can also be included atappropriate concentrations introduced alone or as a mixture with anothersupplement or medium such as a complex nitrogen source.

If mammalian host cells are used to produce neurotrophin, they may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, Meth. Enz., 58: 44 (1979); Barnes andSato, Anal. Biochem., 102:255 (1980); U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 5,122,469; or 4,560,655; WO 90/03430; WO 87/00195;or U.S. Pat. No. Re. 30,985, the disclosures of all of which areincorporated herein by reference, can be used as culture media for thehost cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin TM drug),trace elements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of in vitro mammalian cell cultures can befound in Mammalian Cell Biotechnology: A Practical Approach, M. Butler,ed. (IRL Press at Oxford University Press, Oxford, 1991). The aboveprocess can be employed whether the neurotrophin is producedintracellularly, produced in the periplasmic space, or directly secretedinto the medium.

Typically culture fluid is harvested after a suitable period andstandard identification assays, for example, immunoassays such as ELISAand Western blot analysis, or biological assays, such as PC12 celldifferentiation (Greene, L. A., Trends Neurosci. 7:91 (1986)) areperformed. Assays to determine the kind and extent of the variantsdisclosed herein are known in the art or are provided or cited in theExamples (see, for example, Schmelzer et al. J Neurochem. 59 (5):1675-83 (1992) and Burton et al., J Neurochem. 59(5):1937-45 (1992),which are incorporated herein by reference.

The neurotrophin composition prepared from the cells is preferablysubjected to at least one purification step prior to HIC. Examples ofsuitable purification steps include those that are described herein,including affinity chromatography, other techniques for proteinpurification such as chromatography on silica, chromatography on heparinSepharose, chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromatofocusing, and preparative SDS-PAGE,depending on the neurotrophin to be recovered and starting culture used.

In one embodiment where the neurotrophin is directly secreted into themedium, the medium is separated from the cellular debris bycentrifugation, and the clarified fermentation broth or medium is thenused for purification on silica gel. For the silica chromatography,typically the broth is passed through underivatized silica particlessuch that the neurotrophin polypeptide adheres to the silica particles;the silica particles are washed to remove contaminants; and thepolypeptide is eluted from the silica particles with a buffer comprisingan alcoholic or polar aprotic solvent and an alkaline earth, an alkalimetal, or an inorganic ammonium salt.

In a preferred embodiment of the invention, Macroprep High SCation-Exchange Chromatography is employed to separate neurotrophin fromits variants, as well as for decreasing bulk contaminants. This resincan be used as an early step in the purification of a neurotrophin frommammalian cell culture, preferably for fractionation of the harvestedcell culture medium. In another embodiment Macroprep High SCation-Exchange Chromatography is most preferably used immediately aftera protein refolding step. The very high flow property of this cationexchange column allows the large volume of dilute refolded protein orharvested cell culture medium neurotrophin to be readily concentratedbefore subsequent chromatography steps, such as HIC or SP-Sepharose, byadjusting conditions so that the neurotrophins bind the column.Furthermore, the cation exchange nature of the resin allows removal ofnon-bound bulk proteins and some misprocessed variants andchemically-modified variants (e.g., altered charge, MET37-oxidizedvariant). Most importantly, when misfolded variants or chemicallymodified variants (e.g. misprocessed variants, glycosylation variant (asfrom mammalian cell culture production)) that differ in hydrophobicityfrom native neurotrophin are present, the Macroprep resin allows atleast a partial removal of these hydrophobic variants, substantiallyenriching for the native neurotrophin, as determined herein. While notmeant to be limiting, it is believed that the backbone of the resinsupport contains hydrophobic content that promotes non-specificinteractions between neurotrophins and the resin, which has been takenadvantage of as taught herein. Typically, for an elution buffer of pHfrom about pH 5 to 8, more preferably 6 to 8, a 0 to 3 M TMACconcentration is useful. Sodium acetate, when present to increase theionic strength of the elution buffer, allows use of lower TMACconcentration. Chloride is a preferred substitute for acetate ion.

In one example of the embodiment where the neurotrophin is produced inthe periplasmic space, the culture medium or lysate is centrifuged toremove particulate cell debris. The membrane and soluble proteinfractions can then be separated if necessary. The neurotrophin can thenbe purified from the soluble protein fraction and from the membranefraction of the culture lysate, depending on whether the neurotrophin ismembrane bound, is soluble, or is present in an aggregated form. Theneurotrophin thereafter is solubilized and then subsequently refoldedusing an appropriate buffer. The details for this method of isolationfrom the periplasm to produce refolded protein are described below.

Insoluble, non-native neurotrophin is isolated from the prokaryotic hostcells in a suitable isolation buffer by any appropriate technique, e.g.,one involving exposing the cells to a buffer of suitable ionic strengthto solubilize most host proteins, but in which aggregated neurotrophinis substantially insoluble, and disrupting the cells so as to releasethe inclusion bodies and make them available for recovery by, forexample, centrifugation. This technique is well known, and is described,for example, in U.S. Pat. No. 4,511,503.

Briefly, the cells are suspended in the buffer (typically at pH 5 to 9,preferably about 6 to 8, using an ionic strength of about 0.01 to 2M,preferably 0.1 to 0.2M). Any suitable salt, including sodium chloride,is useful to maintain a sufficient ionic strength value. The cells,while suspended in this buffer, are then disrupted by lysis usingtechniques commonly employed such as, for example, mechanical methods,e.g., a Manton-Gaulin press microfluidizer, a French press, or a sonicoscillator, or by chemical or enzymatic methods.

Examples of chemical or enzymatic methods of cell disruption includespheroplasting, which entails the use of lysozyme to lyse the bacterialwall (Neu et al., Biochem. Biophys. Res. Comm., 17:215 (1964)), andosmotic shock, which involves treatment of viable cells with a solutionof high tonicity and with a cold-water wash of low tonicity to releasethe polypeptides (Neu et al., J. Biol. Chem., 240: 3685-3692 (1965)). Athird method, described in U.S. Pat. No. 4,680,262, involves contactingthe transformed bacterial cells with an effective amount of a loweralkanol having 2 to 4 carbon atoms for a time and at a temperaturesufficient to kill and lyse the cells.

After the cells are disrupted, the suspension is typically centrifugedto pellet the inclusion bodies. In one embodiment, this step is carriedout at about 500 to 15,000 times g, preferably about 12,000 times g, ina standard centrifuge for a sufficient time that depends on volume andcentrifuge design, usually about 10 minutes to 0.5 hours. The resultingpellet contains substantially all of the insoluble polypeptide fraction,but if the cell disruption process is not complete, it may also containintact cells or broken cell fragments. Completeness of cell disruptioncan be assayed by resuspending the pellet in a small amount of the samebuffer solution and examining the suspension with a phase-contrastmicroscope. The presence of broken cell fragments or whole cellsindicates that additional disruption is necessary to remove thefragments or cells and the associated non-refractile polypeptides. Aftersuch further disruption, if required, the suspension is againcentrifuged and the pellet recovered, resuspended, and analyzed. Theprocess is repeated until visual examination reveals the absence ofbroken cell fragments in the pelleted material or until furthertreatment fails to reduce the size of the resulting pellet.

In an alternative embodiment, the neurotrophin is isolated from theperiplasmic space by solubilization in a suitable buffer. This procedurecan be in-situ solubilization involving direct addition of reagents tothe fermentation vessel after the neurotrophin has been producedrecombinantly, thereby avoiding extra steps of harvesting,homogenization, and centrifugation to obtain the neurotrophin. Theremaining particulates can be removed by centrifugation or filtration,or combinations thereof.

If the neurotrophin is being unfolded, the degree of unfolding issuitably determined by chromatography of the non-native neurotrophin,including RP-HPLC. Increasing peak area for the non-native materialindicates how much non-native neurotrophin is present.

Once obtained from the solubilized inclusion bodies or at a later stageof purification, the neurotrophin is suitably refolded into an activeconformation as described below.

If the neurotrophin is not already in soluble form before it is to berefolded, it may be solubilized by incubation in alkaline buffercontaining chaotropic agent and reducing agent in amounts necessary tosubstantially solubilize the neurotrophin. This incubation takes placeunder conditions of neurotrophin concentration, incubation time, andincubation temperature that will allow solubilization of theneurotrophin to occur in the alkaline buffer.

Measurement of the degree of solubilization of the neurotrophin in thebuffer is suitably carried out by turbidity determination, by analyzingneurotrophin fractionation between the supernatant and pellet aftercentrifugation on reduced SDS gels, by protein assay (e.g., the Bio-Radprotein assay kit), or by HPLC.

The pH range of the alkaline buffer for solubilization typically is atleast about 7.5, with the preferred range being about 8-11. Examples ofsuitable buffers that will provide a pH within this latter range includeglycine, CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid),AMP (2-Amino-2-methyl-1-propanol), CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid), CHES(2-[N-Cyclohexylamino]ethanesulfonic acid), and TRIS HCl(Tris[hydroxymethyl]aminomethane hydrochloride). The preferred bufferherein is glycine or CAPSO, preferably at a concentration of about 20mM, at a pH of about 8.5 to 11, preferably about 10-11.

The concentration of neurotrophin in the buffered solution forsolubilization must be such that the neurotrophin will be substantiallysolubilized and partially or fully reduced and denatured. Alternatively,the neurotrophin may be initially insoluble. The exact amount to employwill depend, e.g., on the concentrations and types of other ingredientsin the buffered solution, particularly the type and amount of reducingagent, the type and amount of chaotropic agent, and the pH of thebuffer. For example, the concentration of neurotrophin may be increasedat least three-fold if the concentration of reducing agent, e.g., DTT,is concurrently increased, to maintain a ratio of DTT:neurotrophin offrom about 3:1 to 10:1. It is desirable to produce a more concentratedsolubilized protein solution prior to dilution refolding. Thus, thepreferred concentration of neurotrophin is at least about 30 mg/mL, witha more preferred range of 30-50 mg per mL. For example, neurotrophin maybe solubilized to a concentration of about 30-50 mg/mL in 5M to 7M urea,10 mM DTT and diluted, for example, to about 1 mg/ML for folding.

After the neurotrophin is solubilized, it is placed or diluted into arefolding buffer containing 5-40% (v/v) alcoholic or aprotic solvent, achaotropic agent, and an alkali metal, alkaline earth, or ammonium salt.The buffer can be any buffer for the first buffered solution, withCAPSO, glycine, and CAPS being preferred at pH 8.5-11, particularly at aconcentration of about 20 mM, and most preferably CAPSO and glycine. Theneurotrophin may be diluted with the refolding buffer, preferably atleast five fold, more preferably at least about ten fold. Alternatively,the neurotrophin may be dialyzed against the refolding buffer. Therefolding can be carried out at about 2°-45° C., most preferably about2°-8° C. at least about one hour. The solution optionally also containsa reducing agent and an osmolyte.

The reducing agent is suitably selected from those described above forthe solubilizing step in the concentration range given. Itsconcentration will depend especially on the concentrations of alkalineearth, alkali metal, or ammonium salt, neurotrophin, and solvent.Preferably, the concentration of reducing agent is about 0.5 to 8 mM,more preferably about 0.5-5 mM, even more preferably about 0.5-2 mM. Thepreferred reducing agents are DTT and cysteine.

Oxygen in the refold solution may optionally be depleted by addition ofan inert gas, for example helium or argon, to displace the oxygen.

The optional osmolyte is preferably sucrose (in a concentration of about0.25-1M) or glycerol (in a concentration of about 1-4M). Morepreferably, the sucrose concentration is at about 1M and the glycerolconcentration is at about 4M.

The initial concentration of neurotrophin in the folding buffer is suchthat the ratio of correctly folded to misfolded conformer recovered willbe maximized, as determined by HPLC, RIA, or bioassay. The preferredconcentration of neurotrophin (resulting in the maximum yield ofcorrectly folded conformer) is in the range of about 0.1 to 15 mg/mL,more preferably about 0.1 to 6 mg/mL, and most preferably about 0.2 to 5mg/mL.

The degree of refolding that occurs upon this incubation is suitablydetermined by the RIA titer of the neurotrophin or by HPLC analysis withincreasing RIA titer or correctly folded neurotrophin peak size directlycorrelating with increasing amounts of correctly folded, biologicallyactive neurotrophin conformer present in the buffer. The incubation iscarried out to maximize the yield of correctly folded neurotrophinconformer and the ratio of correctly folded neurotrophin conformer tomisfolded neurotrophin conformer recovered, as determined by RIA orHPLC, and to minimize the yield of multimeric, associated neurotrophinas determined by mass balance. Alternatively, the species can bedetermined via the methods provided below and in the Examples. Guanidineis a preferred denaturing agent for refolding.

After the neurotrophin is refolded, the following procedures as taughtherein, individually or in combination, are exemplary of suitablepurification procedures for obtaining greater purity and homogeneity:fractionation on cation-exchange columns; hydrophobic interactionchromatography (HIC); and chromatography on silica.

Whether a refolding step is part of the process or not, a preferred stepfor separation of a neurotrophin from its variants is separation onhydrophobic interaction chromatography resin. During fermentation,purification or in vitro protein refolding, some protein can bechemically modified, misprocessed, or may not refold to its nativethree-dimensional structure but rather to other structures which differwith respect to their stability, solubility, immunogenicity, orbioactivity. These variants must be removed during recovery to avoidundesirable side-effects such as antigenicity or loss of potency. If thevariant is insoluble it can be easily removed by solid/liquid separationtechniques such as centrifugation and filtration. However, if thevariant is soluble, higher resolution adsorption techniques such aschromatography will be required to remove them. When produced inprokaryotic cells or when refolded in vitro, neurotrophins form asoluble stable misfolded variant. Misfolded neurotrophin has an altereddisulfide pairing pattern and three-dimensional structure relative tonative neurotrophin and lacks native pharmacological activity. Whenproduced in eucaryotic cell culture, e.g. mammalian cell culture, thevariant forms are typically misprocessed forms. These also typicallylack native pharmacological activity and should be removed. HIC has beenfound herein as suitable for separating these variants from nativeneurotrophin.

HIC involves sequential adsorption and desorption of protein from solidmatrices mediated through non-covalent hydrophobic bonding. Generally,sample molecules in a high salt buffer are loaded on the HIC column. Thesalt in the buffer interacts with water molecules to reduce thesalvation of the molecules in solution, thereby exposing hydrophobicregions in the sample molecules which are consequently adsorbed by theHIC column. The more hydrophobic the molecule, the less salt needed topromote binding. Usually, a decreasing salt gradient is used to elutesamples from the column. As the ionic strength decreases, the exposureof the hydrophilic regions of the molecules increases and moleculeselute from the column in order of increasing hydrophobicity. Sampleelution has also be achieved by the addition of mild organic modifiersor detergents to the elution buffer. HIC is reviewed in ProteinPurification, 2d Ed., Springer-Verlag, N.Y., pgs 176-179 (1988).

The strength of the association between a protein and a matrix dependson several factors, including the size and hydrophobic character of theimmobilized functional group, the polarity and surface tension of thesurrounding solvent, and the hydrophobicity of the protein. The bindingcapacity of HIC matrices tends to be low due to the need for theimmobilized hydrophobic ligand to be widely spaced. Further, thecapacity of a medium for a given protein varies inversely with the levelof hydrophobic impurities in the sample preparation. In order to resolvea desired protein from variants and other impurities whilesimultaneously maximizing capacity, it is necessary to identify asuitable HIC solid-phase medium as well as suitable mobile phases forload, wash, and elution.

As determined herein, the most suitable media for separating correctlyfolded and misfolded neurotrophins or misprocessed forms from intact,correctly processed forms, were those having immobilized phenylfunctional groups. Phenyl-based HIC media from different vendorsexhibited different efficiency for resolving these neurotrophin forms.Best results were achieved with Phenyl Toyopearl media by TosoHaas andPhenyl Sepharose Fast Flow Low Sub (low substitution). TSK Phenyl 5PWwas also suitable. Other HIC-immobilized functional groups can functionto separate these forms. Examples were octyl groups, such as those onOctyl Sepharose CL4B media from Pharmacia, and propyl groups, such asthose on High Propyl media from Baker. Less preferred are the alkoxy,butyl, and isoamyl functional group resins.

HIC was useful for separation of neurotrophins from their variants inmammalian cell culture. For example, as was determined herein,rhNGF-expressing-CHO cell culture contained incorrectly proteolyticallyprocessed variants, such as those in which a partial precursor sequenceis present, e.g., precursor NGF, hybrid precursor NGF, and clippedprecursor NGF sequences. Also found in the mammalian cell culture mediumwere glycosylated NGF and glycosylated forms of the incorrectlyproteolytically processed variants. Undesirable glycosylated forms,which in the case of NGF can be seen as a higher molecular weightspecies (+2000 kD), could generate an unwanted antigenic response in apatient and contribute to poor product quality or activity. HICeffectively separated hydrophobic variants, primarilyN-terminal-proteolytically-misprocessed variants, including glycosylatedforms, from rhNGF. As shown in the examples, theprecursor-sequence-containing and clipped precursor sequence NGF and theglycosylated forms of both NGF and the precursor-sequence-containing NGFeluted in the leading edge of the NGF peak during phenyl-HIC. Thus, arhNGF composition could be obtained that was substantially free of thesespecies, and that was particularly suited for a subsequent step such ashigh performance cation-exchange chromatography. HIC is applicable toother neurotrophins, as well as NGF, regardless of source. For example,HIC is useful to separate NGF monomers from dimers, either homo- orhetero-dimers depending on the monomer forms present, as well asdistinguish dimer forms which also differ in hydrophobicity, that areobtained after in vitro refolding or when produced and secreted frommammalian cells. A preferred source of neurotrophin mixtures for usewith HIC is mammalian cell culture, more preferably CHO cell culture.The culture is preferably subjected to at least one prior purificationstep as discussed herein. HIC is particularly effective in separatingmisprocessed glycosylated variant(s) from the native recombinantneurotrophin. In the case of rhNGF, the glycosylated and preproNGF formsare less hydrophobic than native NGF, thereby eluting before native NGF.Misfolded forms of neurotrophins (when bacterially produced) are alsomore hydrophobic, eluting earlier than the native neurotrophin.

The most preferred HIC resin for separating neurotrophin forms werethose having immobilized phenyl functional groups. Phenyi-based HICmedia from different vendors exhibited different efficiency forresolving these NGF forms. Of the phenyl-HIC resins, Phenyl Toyopearlmedia by TosoHaas is most preferred and Phenyl Sepharose Fast Flow LowSub (low substitution) and TSK Phenyl 5PW are preferred. Preferred HICfunctional groups include the alkoxy, butyl, and isoamyl moieties.

Using HIC, a variety of mobile phase conditions can be used to wash anddifferentially elute neurotrophin forms. These mobile phases can containseveral different chemical species that influence the associationbetween a neurotrophin and the stationary phase in different ways.Correctly folded and misfolded neurotrophin, e.g. NT-4/5, can beresolved on a HIC column by decreasing salt gradients or step-wisedecrease, for example of mobile-phase salt, e.g., ammonium sulfate, NaClconcentration, acetate concentration. Salts can influence the binding ofa neurotrophin to the resin by modulating the surface tension of themobile phase. Other agents that affected surface tension were sodiumcitrate and tetramethyl ammonium chloride, as discussed in the Examples.Variants can also be resolved during column chromatography by elutingbound protein with increasing gradients or step-wise increase inconcentration of relatively polar organic solvents. Examples of suitablesolvents include ethanol, acetonitrile, and propanol. The strength ofthe association between neurotrophin forms and HIC resin also dependedon the mobile-phase pH, with neutral conditions preferred. The relativehydrophobicity of correctly folded and misfolded neurotrophin alsodepended on solution pH. Separation of variants from native neurotrophincould also be obtained by simultaneously varying several properties ofthe mobile phase during gradient or stepwise elution. For example, amobile phase that simultaneously varied in salt concentration and apolarsolvent concentration during elution provided resolution better thanwhen only salt was varied.

For HIC, salts discussed herein, including ammonium sulfate, citrate,acetate, and potassium chloride can be used. Depending on the salt used,the salt concentration is typically 0.5 M to 3 M, more preferably 0.5 to2.5M, to achieve binding of neurotrophin to the resin. For example, abinding buffer of 0.8 to 1.5 M salt is preferred for NGF, with highersalt concentrations leading to precipitation of NGF onto the resinresulting in lower recovery. For NT-3 a binding buffer at pH 7 with asalt concentration of 1.0 to 2.5 is preferred, with 1.25 to 1.75 M NaClbeing more preferred, and 1.5 M most preferred. For NT-4/5 a bindingbuffer at pH 7 with 1 to 3 M salt is preferred, with 2 to 2.75 M beingmore preferred, and 2.5 M NaCl being most preferred. In the case ofNT-4/5, when 2.5 M NaCl was preferred for loading, 2M NaCl was preferredfor elution with organic solvent present (e.g. 10% alcohol, pH 7).Preferably, a lowering of the salt concentration is used to elute andseparate a neurotrophin and its variants. In order to achieve elution,the salt concentration in the elution buffer is typically lower thanthat in the loading buffer, but it can be the same concentration whencompensated for with organic solvent.

In addition, the use of organic solvent has another advantage, as hasbeen found herein, that the addition of an organic solvent improves theelution pattern by resulting in narrower peak profiles. In addition toethanol, other organic solvents discussed herein can be used, includingpropanol, isopropanol, and lower alkylene glycols, such as propyleneglycol, ethylene glycol and hexylene glycol. The organic solvent at 5 to25% (v/v), more preferably 5 to 20% (v/v), will typically elute acorrectly folded neurotrophin. The elution with organic solvent can beeither gradient or step-wise. The pH range is preferably near neutral toslightly acidic, from pH 5 to 8, more preferably pH 6 to 8, pH 6.5 to7.5, and most preferably pH 7. Any of the buffers discussed herein,including MOPSO, MOPS, HEPES, phosphate, citrate, ammonium, acetate, canbe used as long as they buffer at the desired pH.

In view of the discovery by the present inventors of certain undesirableneurotrophin variants arising from recombinant production of aneurotrophin, as reported herein, the use of high performancecation-exchange chromatography, preferably in preparative mode, allowsseparation of the charge-modified variants, such as carbamylated,oxidized, isoasp, deamidated, and certain clipped forms (e.g. C-terminaltruncated forms of NGF) from native neurotrophin. For example,N-terminal clipped forms (e.g., 2 to 4 N-terminal amino acid deletions)that result in charge alteration, which may occur during bacterialfermentation as in the case of NT-4/5 and NT-3, can now be removed. Inthe case of neurotrophins produced in mammalian cell culture, C-terminaltruncation may occur in the highly charged terminal region. For example,118 form of NGF may be niisprocessed or cleaved at its C-terminus to117, 114 and 115 forms. These can be separated from native 118 NGF byhigh performance cation exchange chromatography. Particularly preferredembodiments use SP-Sepharose High Performance, Fractogel EMD SO3, orpolyaspartic acid resin, of which PolyCAT A is particularly preferred.Most preferably, at large scale, SP-Sepharose High Performance orFractogel EMD SO3 resins are used.

Compositions obtained by the processes described herein will besubstantially pure neurotrophin, more usually and preferably essentiallypure, and will be substantially free of neurotrophin variants, morepreferably essentially free of neurotrophin variants. For example, atypical SP-Sepharose pool after purification of NGF from CHO cellculture, contains about 92% 118, 4.6% 120, 1% deamidated NGF, 1%oxidized NGF, and 1%isoasp NGF. Routinely the amount of each speciesranges from about 85 to 93% for 118, 0 to 5% for 120 (depending in largepart on the extent of endogenous and/or exogenous proteolysis that isused), 0 to 5% for 117, 0 to 3% for deamidated forms, 0-2% for isoaspforms, and 0 to 2% for oxidized forms. The purity of NGF (all species)is routinely greater than 99.5%.

After the neurotrophin is eluted from the column, it is suitablyformulated into a composition with a carrier, preferably apharmaceutical composition with a physiologically acceptable carrier.Neurotrophin compositions are preferably sterile. The neurotrophincompositions of the invention also find use in in vitro, for example, topromote growth and survival of neurons in culture.

The chemical and physical stability of recombinant human nerve growthfactor (NGF) in aqueous solution was investigated between 5 and 37° C.,in the pH range 4.2 to 5.8. NGF chemical stability increased withincreasing pH. In succinate buffer at pH 5.8, NGF physical stabilitydecreased due to protein aggregation. Based on both the 5° C. stabilitydata and accelerated degradation studies at 37° C., the optimalformulation was found to be acetate buffer at pH 5.5. (see WO 97/17087which is incorporated herein by reference) Reversed-phase HPLC was theprimary stability indicating method, showing conversion of Asn-93 toiso-Asp to be the primary degradation pathway at 5° C. Quantitation ofNGF degradation by cation exchange chromatography was complicated by therearrangement of the NGF monomer variants into various mixed dimers overtime (dimer exchange). Treatment of samples and controls with diluteacid rapidly equilibrated the monomer distribution in the dimers,allowing NGF degradation to be quantitated in the absence of dimerexchange. Benzyl alcohol and phenol were evaluated for theircompatibility and stability with rhNGF in two liquid formulations formulti-use purposes. These two formulations consist of 0.1 mg/ml, proteinin 20 mM sodium acetate at pH 15.5 and 136 mM sodium chloride with andwithout 0.01% pluronic acid (F68) as surfactant. The finalconcentrations of benzyl alcohol and phenol in each of these twoformulations were 0.9 and 0.25%, respectively. Based on the 12 monthstability data, rhNGF is more stable with benzyl alcohol than phenol inthese formulations. Benzyl alcohol preserved rhNGF formulation with thepresence of surfactant is as stable as the formulation with nosurfactant added, indicating that the addition of F68 to rhNGFmulti-dose formulation is not required for stability purpose. Therefore,a formulation consisting of 0.1 mg/mL protein in 20 mM acetate, 136 mMNaCl, 0.9% benzyl alcohol, pH 5.5 is recommended for rhNGF used formultiple dosing in Phase III clinical trails. This rhNGF multi-doseformulation passed the USP and EP preservative efficacy test after 6months at 5° C., and is as stable as the current liquid formulation at 2mg/mL. However, the formulation should avoid exposure to intensive lightdue to the presence of benzyl alcohol as preservative which is lightsensitive.

In general, the compositions may contain other components in amountspreferably not detracting from the preparation of stable, liquid orlyophilizable forms and in amounts suitable for effective, safepharmaceutical administration.

Neurotrophin is formulated with a pharmaceutically acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious topolypeptides. This formulation step is achieved by desalting ordiafiltering using standard technology.

Generally, the formulations are prepared by contacting the neurotrophinuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, trehalose,glucose, mannose, or dextrins; chelating agents such as EDTA, sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.The final preparation may be a liquid or lyophilized solid.

Neurotrophin to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticneurotrophin compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle. The aboveformulations are also suitable for in vitro uses.

Neurotrophin ordinarily will be stored in unit or multi-dose containers,for example, sealed ampules or vials, as an aqueous solution, or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-mL vials are filled with 5 mL ofsterile-filtered 1% (w/v) aqueous neurotrophin solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized neurotrophin using bacteriostaticWater-for-Injection.

A therapeutically effective dose of an neurotrophin formulation isadministered to a patient. By “therapeutically effective dose” herein ismeant a dose that produces the effects for which it is administered. Theexact dose will depend on the disorder to be treated, and will beascertainable by one skilled in the art using known techniques. Ingeneral, the neurotrophin formulations of the present invention areadministered at about 0.01 .mu.g/kg to about 100 mg/kg per day.Preferably, from 0.1 to 0.3 ug/kg. In addition, as is known in the art,adjustments for age as well as the body weight, general health, sex,diet, time of administration, drug interaction and the severity of thedisease may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. Typically, the clinicianwill administer neurotrophin formulations of the invention until adosage is reached that repairs, maintains, and, optimally, reestablishesneuron function. The progress of this therapy is easily monitored byconventional assays.

Neurotrophin optionally is combined with or administered in concert withother neurotrophic factors including NGF, NT-4/5, NT-3, and/or BDNF andis used with other conventional therapies for nerve disorders.

In the case of NGF, preferably a composition comprises apharmaceutically effective amount of nerve growth factor and apharmaceutically acceptable acetate-containing buffer. The compositioncan have a pH from pH 5 to 6. The buffer is preferably sodium acetate.The acetate concentration is preferably 0.1 to 200 mM. The compositionpreferably has an NGF concentration of 0.07 to 20 mg/ml. And, thecomposition optionally further contains a pharmaceutically acceptablepreservative, such as benzyl alcohol, phenol, m-cresol, methylparaben,or propylparaben. Preferably the preservative is benzyl alcohol. Thebenzyl alcohol concentration is preferably from 0.1 to 2.0%. Thecomposition can optionally contain a pharmaceutically acceptablesurfactant. And, the composition can optionally, but preferably, containa physiologically acceptable concentration of sodium chloride. A morepreferred composition contains nerve growth factor at a concentration ofat least about 0.1 mg/ml and an acetate ion concentration of 10 mM to 50mM. Even more preferably, the composition contains nerve growth factorat a concentration of 0.1 to about 2.0 mg/ml and acetate ion at aconcentration of 10 mM to 50 mM. A most preferred composition containsNGF at a concentration of 0.1 mg/ml, sodium acetate concentration of 20nM, pH 5.5, sodium chloride concentration of 136 mM, and benzyl alcoholat 0.9% (v/v).

Another embodiment contains an NGF concentration of 2.0 mg/ml. a sodiumacetate concentration of 10 mM, pH 5.5, and a sodium chlorideconcentration of 142 mM. It is preferable to formulate NGF with 0.1mg/ml, 20 mM sodium acetate, 136 mM sodium chloride, 0.9% (v/v) benzylalcohol, at a pH of 5.5. As discussed herein, the 118/118 homodimer is apreferred form of NGF. NGF is purified at about pH 6 to 8 to maintainthe normal dimer form. However, the percentage of (proteolytically)clipped forms represents monomeric forms, which become apparent and canbe determined by reversed-phase HPLC. The acid conditions of theanalytical HPLC dissociates the dimers. The existence of differentdimeric forms of NGF—120/120, 120/118, 118/118, etc.—has been published(Schmelzer et al. J. Neurochem. 59:1675-1683 (1992), which isspecifically incorporated herein in its entirety, mainly for itsanalytical and bioassays that were used in the current studies as wellas for its general teachings). That publication reported that the invitro activities were the same for each dimeric form. However, incontrast, the present studies herein demonstrate for the first time thatthe 120/120 dimer is less active, about 80-90% as active, as the 118/118species, using a radioreceptor based assay. In one form of the assay,rat PC-12 cells membranes are isolated and used for competitive bindingbetween NGF standard and the various test species. The RRA has both P75and trkA receptors. It was also found herein that the 117/117 species isas active as the 118/118 species. Furthermore, use herein of a PC-12based assay confirmed the receptor-based assay finding, showing that the120/120 form is about 60% as active as the 118/118 form. Alsoincorporated in its entirety specifically by reference is Burton et al,J. Neurochem. 59:1937-1945 (1992) mainly for its analytical and bioassaythat were used in the current studies as well as for its generalteachings.

The 118/118 form is believed to be more bioavailable in human patientsthan the 120/120 form. The increase in bioavailability is at least 4 to5 fold. This difference is significant, surprising, and unexpected inview of the art.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLES Example 1 Purification of 118/118 NGF Homodimer

This example illustrates purification of NGF and the rationale for eachstep. As in each of the Examples, one skilled in the art can readilydetermine and adjust column dimensions and flow rates to compensate forinitial culture volumes and protein concentrations as is well-known inthe art.

Harvested Cell Culture Fluid

Recombinant CHO cell was transfected with an expression vectorcontaining the 120 amino acid human NGF encoding DNA sequence. Topromote secretion and processing the NGF prepro sequence was alsopresent. After culturing of the recombinant CHO cells, the cell culturemedium was harvested. The Harvested Cell Culture Fluid (HCCF) containedNGF species 120, 118, and 117. About 40-70% of the NGF was typically a118/118 homodimer with the remainder as heterodimers 120/118, 120/120,and a small amount as 118/117. As taught herein, these species can beseparated by the SP-Sepharose HP column.

Harvested Cell Culture Fluid was concentrated approximately 20-foldusing Millipore 10 Kd cutoff membranes (either cellulose, composite orpolysulfone were used interchangeably). To the concentrate was added 0.1volumes of 1.0 M Tris, pH 8.2. The diluted material was microfilteredusing a 0.22 um filter and transferred to a holding tank at 37 degreesC. for 2 to 18 hours. Conversion of 120/120 form to the 118/118 form iscatalyzed by an endogenous protease during holding.

Silica Gel Chromatography

The microfiltrate was adjusted to 1M NaCl and applied to a Silica GelColumn equilibrated in 1M NaCl, 25 mM MOPSO, pH 7. The column was washedwith 1M NaCl, 25 mM MOPSO, pH7. Suitable pH range is about pH 6 to 8,with a preferred pH of 7. The column was then washed with 25 mM MOPSO,pH 7. A low conductivity wash removes host cell proteins. Bound NGF waseluted with 50 mM MOPSO, 0.5 MT MAC, 20% reagent anhydrous grade alcohol(94-96% Specially Denatured alcohol formula 3A (5 volumes of methanoland 100 volumes of 200 proof ethanol) and 4-6% isopropanol). Otheralcohols can be used such as 20% propanol, 20% isopropanol and 20%methanol. As used herein, “alcohols” and “alcoholic solvents” are meantin the sense of the commonly used terminology for alcohol, preferablyalcohols with 1 to 10 carbon atoms, more preferably methanol, ethanol,iso-propanol, n-propanol, or t-butanol, and most preferably ethanol oriso-propanol. Such alcohols are solvents that, when added to aqueoussolution, increase the hydrophobicity of the solution by decreasingsolution polarity. Ethanol is most preferred. The lower limit of alcoholis whatever percentage that elutes and the upper limit is set by theneed to avoid protein denaturation. The solvent is preferably 5% to 25%,more preferably 5 to 20%, even more preferably 5 to 15%. TMAC istetramethyl ammonium chloride, which is present to elute NGF. TMAC canrange from 0.1 to 1 M. With the range 0.3 to 0.7 M being more preferred.The amount of TMAC used to elute NGF is a function of pH and alcoholconcentration. The lower the pH the less amounts of alcohol and TMAC isrequired. The pH can be between about pH 4 to 8. In this example thepreferred pH was 7, which allows very minimal adjustment of the pooledfractions prior to loading onto the next column. The upper pH limit isdetermined by the pH necessary to load the next column, and the lowerlimit by that useful to elute NGF efficiently.

S-Sepharose Fast Flow Chromatograph

The eluant containing NGF was pooled, diluted to a conductivity of lessthan 15.5 ms/cm with purified water, and pH adjusted to 7.0. Thematerial was held no longer than 8 hours since several proteases werestill present; however, no activity of the endogenous protease thatconverts 120 amino acid NGF to 118 form was observed. The material wasapplied to an S-Sepharose Fast Flow chromatography column (acation-exchange resin S-SEPHAROSE TM agarose Fast Flow TM (Pharmacia))equilibrated in 25 mM MOPSO, pH 7. The column is washed with 25 mMMOPSO, pH 7. A suitable pH range is from about pH 6 to 8, with pH 7preferred. The column was then washed with 0.16 M NaCl, pH 7. The boundNGF was eluted with 0.5 M NaCl, pH 7. The elution salt molarity canrange from 0.3 to 1.0 M, more preferably 0.4 to 0.6 M. The lower limitis set by the usefulness to elute all the NGF, and the upper limit isset by the need to avoid removing contaminants and causing hydrophobicinteractions on the column which would interfere with elution of NGF.Other salts can be used, KCl being a preferred alternative. Elution with0.5 M NaCl, pH 7, is preferred in order to obtain a pool with a smallvolume. At higher salt concentrations, e.g., over 1 M, tightly boundcontaminants may elute.

Phenyl Toyopearl 650M Chromatography

SSFF column fractions containing NGF were pooled, adjusted to 1 M NaCl,and applied to a Phenyl Toyopearl 650M column. The column was washedwith 25 mM MOPSO, pH 7. A suitable pH is in the range of about pH 5 to8. The bound NGF was eluted with a 10 CV (column volume) linear gradientbeginning with gradient buffer A (25 mM MOPSO, pH 7, 1.0 M NaCl) andending with gradient buffer B (20% alcohol in 80% 25 mM MOPSO, pH 7).Fractions containing NGF were analyzed by SDS-PAGE polyacrylamide gelelectrophoresis to determine which fractions harbored the precursor NGFspecies. Fractions containing NGF and were selected and pooled to removeprimarily incorrectly processed variants, such as those in which apartial precursor sequence is present, e.g., precursor NGF, hybridprecursor NGF, and clipped precursor NGF sequences, to obtain an NGFcomposition substantially free of any NGF precursor sequences. Thephenyl column also removed the small amount of glycosylated NGF andglycosylated NGF precursor sequences. The precursor and clippedprecursor NGF sequences along with the glycosylated forms of both NGFand precursor NGF eluted in the leading edge of the NGF peak. Thus, thiscolumn readily separated NGF from various NGF species to obtain an NGFcomposition substantially free of these species. In this step variantNGF hydrophobic variants, primarily misprocessed variants, includingproteolytic and glycosylated variants, were separated using HIC.

The most suitable media for separating NGF forms were those havingimmobilized phenyl functional groups. Phenyl-based HIC media fromdifferent vendors exhibited different efficiency for resolving these NGFforms. Best results were achieved with Phenyl Toyopearl media byTosoHaas. HIC resins Phenyl Sepharose Fast Flow Low Sub (lowsubstitution) and TSK Phenyl 5PW worked well. Other HIC functionalgroups were less suitable, and less effective, under these conditions,including the alkoxy, butyl, and isoamyl moieties.

The PhenylToyopearl pool contained 75% 118, 10% 120, 7% 117, 1.8%deamidated NGF, 1.4% oxidized NGF, and 2.0% isoasp NGF, with theremaining 2.8% being other unidentified NGF species.

Optionally, the pooled fractions were acid treated to achieve viralinactivation at a pH less than 3.95 for a minimum of 15 minutes.

SP-Sepharose HP Chromatography

The HIC pool was diluted with 0.5 to 1 volumes of water and the dilutedpool was adjusted to pH 6. The pool was loaded onto an SP-Sepharose HPcolumn equilibrated with 0.2 M sodium chloride, 20 mM succinate, pH 6,containing 5% reagent alcohol (as in the Silica gel column step). Thecolumn was washed with 0.2 M NaCl, 20 mM succinate, pH 6 containing 5%reagent alcohol (Formula SDA-3A alcohol; the alcohol is optionallypresent). Alcohol helps reduce non-specific (mostly hydrophobic)interactions of NGF with the resin backbone. A suitable alcohol range isfrom about 0 to 10%. The loading pH is from about pH 5 to 8, which ischosen to achieve and maintain maximal stability of NGF and separationof NGF variants. The column was washed with two column volumes ofequilibration buffer. Bound NGF was eluted and separated from variantsby a linear 22-column-volumes gradient by mixing of 11 column volumes ofgradient buffer A (0.25 M NaCl, 0.02 M succinate, pH6, containing 5%alcohol) and 11 column volumes of gradient buffer B (0.5 M NaCl, pH 6).The alcohol is optional. The 118/118 NGF typically eluted at 0.35 M to0.40 M NaCl concentration.

The column fractions were analyzed for NGF and variant NGF content.Fractions are preferably analyzed by C4 RP-HPLC as described bySchmelzer et al. (1992), supra., and Burton et al. (1992), supra.Fractions were selected and pooled to obtain a composition of NGF thatwas substantially free of modified NGF variants, e.g., charged speciessuch as oxidized, deamidated and isoasp NGF species. The previous HICcolumn cannot effectively remove other variant forms of NGF such asoxidized and isoasp NGF. The HIC column does effectively removemisfolded proteins and glycosylated, which bind tighter to the HIC resinthan correctly folded NGF, since they tend to be more hydrophobic.Accordingly, the cation exchange resin, e.g., SP-Sepharose HP, was usedto remove altered charge variants not removed by the HIC resin.

The SP-Sepharose pool typically contained about 92% 118, 4.6% 120, 1%deamidated NGF, 1% oxidized NGF, and 1% isoasp NGF. Routinely the amountof each species ranges from about 85 to 93% for 118, 0 to 5% for 120, 0to 5% for 117, 0 to 3% for deamidated forms, 0-2% for isoasp forms, and0 to 2% for oxidized forms. The purity of NGF (all species) is routinelygreater than 99.5%.

Formulation

The SP-Sepharose HP pool was prepared for formulation byultrafiltration/diafiltration into a formulation buffer. An acidicbuffer is preferably used, preferably acetate at pH 5 as discussedabove. The 118/118 NGF composition is substantially free of NGF variantsand is substantially pure NGF. The formulated material is suitable fortreating neuronal disorders, particularly peripheral neuropathyassociated with diabetes and peripheral sensory neuropathy associatedwith AIDS.

Example II Purification of the 120/120 NGF Homodimer at Large ScaleHarvested Cell Culture Fluid

HCCF was obtained from a 12,000 liter CHO cell culture generally asdescribed in Example I. The NGF species distribution in the HCCF wasabout 40-65% 120/120 homodimer with 120/118 heterodimer with theremaining as 118/118 homodimer. The medium was typically quicklyprocessed to minimize proteolytic conversion of 120 to 118.

Macroprep High S Cation-Exchange Chromatography

The HCCF was loaded onto a Macroprep High S Cation-ExchangeChromatography column, washed with 1.5 M sodium acetate, 50 mM HEPES pH7. Bound NGF was eluted with 1.5 M NaCl, 0.25 M TMAC, 0.2% thiodiglycol,pH 7. The Macroprep column can be run at pH 5 to 8 with adjustment ofthe acetate concentration. Chloride is a preferred substitute foracetate ion. The NGF eluted due to the TMAC gradient. TMAC is a salthaving both ionic and hydrophobic character, which is a useful propertysince the backbone support of some resins contains hydrophobic contentthat promotes non-specific interactions between NGF and the resin.Typically, for an elution buffer of pH from about pH 6 to 8, a 0-3 MTMAC concentration is useful. Fractions containing NGF were pooled.

Silica Gel Chromatography

The pool was directly applied to a Silica Gel Chromatography Column.Silica provides a mixed mode chromatography resin, having ionic,polarity, and hydrophobic interactions that play a part in proteinbinding characteristics. The column was equilibrated in 1M NaCl, 25 mMMOPSO, pH 7. The column was washed with 1M NaCl, 25 mM MOPSO, pH7(preferably about pH 5.0 to 8.5, more preferably pH 6 to 8, and mostpreferably pH 7). Bound NGF was eluted with 25 mM succinate, pH 3.9, 50mM TEAC (tetraethylammonium chloride). TEAC is a more powerful eluantthan TMAC. The pH preferably ranges from about pH 3.5 to 8. However, apH above 7.5 for extended periods of time should be avoided in order toprevent or reduce formation of the deamidated NGF species. Generally,the lower the pH of the buffer, the lower the concentration of themixed-property salt, e.g., TMAC or TEAC, required to elute NGF from theSilica column. Buffers having a good buffering capacity near pH 4 to 5are suitable for use. The presence of salt in the elution buffer isoptional, such that the column can be washed with a MOPSO buffer withoutsalt prior to application of the elution buffer.

Phenyl Sepharose Fast Flow Chromatography

The fractions containing NGF were identified and pooled. The pool wasadjusted to 0.7 M acetate, pH 7, 25 mM MOPSO. The adjusted pool wasloaded onto a Phenyl Sepharose Fast Flow Chromatography columnequilibrated with gradient buffer A (0.7 M acetate, 25 mM MOPSO, pH 7).The column was washed with a gradient from 90% gradient buffer A (0.7 Macetate, 25 mM MOPSO, pH 7) to 10% gradient buffer B (25 mM MOPSO, pH 7,20% propylene glycol). Other glycols can be substituted, such ashexylene glycol. Typically the wash was about 2 to 3 CV or until astable baseline OD is achieved. The wash removed some host cellproteins. Bound NGF was eluted with a linear 10 CV gradient from a mixof 90% gradient buffer A and 10% gradient buffer B to a mix of 10%gradient buffer A and 90% gradient buffer B. NaCl or sodium sulfate cansubstitute for acetate in the HIC buffers. The pH is preferably fromabout pH 5 to 8, more preferably about 5.5 to 7.5, with pH 6 to 8acceptable, and most preferably about 7. The column separated anyremaining precursor sequences, partial precursor sequences, orglycosylated forms, present as a homodimer or as a heterodimer of amature NGF monomer and a NGF monomer that still has part of theprecursor sequence present. The precursor and glycosylated forms of NGFare present in the leading edge of the elution peak, such thatNGF-containing fractions were pooled to substantially exclude thesespecies.

The HIC pool contained about 72% 120 monomer, 17% 118 monomer, 2.8% 117monomer, 3.6% R120 monomer, 0.8% isoasp forms, 1.3% oxidized forms, and1% deamidated forms, as separated and detected on an analytical HPLCsystem.

SP-Sepharose HP Chromatograph

Fractions containing NGF from the HIC step were pooled. At large scalethis was accomplished by directing the column effluent at theappropriate time to a pool tank (holding tank). The pH of the pool wasadjusted to pH 6, and applied to a SP-Sepharose HP Chromatographycolumn. The column was washed with 20 mM succinate, 0.2 M NaCl, pH 6(gradient buffer A). The bound NGF was eluted with a 22 CV gradientbeginning with a mixture of 70% gradient buffer A and 30% gradientbuffer B to a final mixture of 80% gradient buffer B (0.7 M NaCI/pH 6)and 20% gradient buffer A. The pH is preferably from pH 5 to 8, morepreferably pH 5.7 to 6.5, and most preferably pH 6. A representativechromatogram is shown in FIG. 1.

The SP-Sepharose HP pool routinely contained about 95% 120 form, 3% R120form, 0.65% isoasp form, 0.6% oxidized form, 0.6% deamidated form. Otherunidentified forms of NGF were at about 0.6%, and comprised ofdi-oxidized NGF (Met37 and Met 92) with a deamidated Asn45 present. AnHPIEX analysis comparing a representative HIC pool (loaded onto theSP-Sepharose resin) and the pool obtained after SP-Sepharosechromatography is shown in FIG. 2. Each of the three major clipped formsof NGF, 120, 118, and 117, may have variants, but the variants, such asoxidized and isoasp forms, of the predominant clipped form during apurification (120 in this example) may mask analysis of the variantsfrom the less predominant forms (118 and 117 in this example). An HPLCanalysis of fractions from a representative run is shown in FIG. 3.

The SP-Sepharose HP effectively removed variants present in the HICpool. The R120 form has an additional arginine residue at the N-terminusof NGF; usually the N-terminal amino acid sequence of rhNGF is SSSHP,but R120 has an N-terminal sequence of RSSSHP. Thus the R120 form ismore basic than mature NGF and was separated by SP-SHP. It also haslower bioactivity, probably related to the fact that the NGF N-terminalis necessary for receptor (trkA) binding. The oxidized NGF form is amono-oxidized form having the methionine at position 37 oxidized,yielding a more acidic form that elutes on the leading edge of the NGFpeak. The isoasp form contains a modification of the aspartic acid atamino acid 93. The isoasp form is slightly more basic and thus bindsslightly tighter to the SP-Sepharose HP resin. NGF species containingisoAsp93 eluted in the trailing edge of the elution peak. Deamidationoccurs at asparagine residues, typically at asparagine at position 45.NGF containing deamidated Asn, which yields an Asp at position 45, isslightly more acidic, appearing at the leading edge of the elution peak.

Fractogel EMD SO3 is a less preferred alternative resin to SP-SepharoseHP resin for separating charged variants of the NGF species. When usingthis less preferred resin, higher concentrations of NaCl are needed toelute NGF.

Formulation

The bulk material was formulated by UF/DF into formulation buffer as inthe prior Example. In the final bulk product, the 120 form rangedroutinely from about 92 to 97%, the R120 from about 1 to 4%, the isoaspform from about 0.2 to 1.5%, the oxidized form from about 0.2 to 2%, andthe deamidated form from about 0.2 to 2%. The 117 and 118 forms wereroutinely less than about 2%. The final bulk product was routinely atleast 99.5% pure NGF (including all species).

Example III Isolation of 118/118

In one preferred embodiment to obtain a substantially pure 118/118 NGFcomposition that is substantially free of NGF variants, the method ofExample It was followed with the following modifications. An immobilizedtrypsin column is used between the Macroprep High S column and theSilica column. The Macroprep pool is directly loaded onto theimmobilized trypsin column, after adjusting the pH to between about pH 5to 8.5, most typically 6.5 to 7.5, if necessary. The pool was passedthrough the column during which time most of the NGF was converted tothe 118 form. The protease digestion converts the 120 form to the 118form by cleavage of the C-terminal VRRA to VR. To achieve the limitedand selective cleavage, a trypsin or trypsin-like protease is used,preferably trypsin, more preferably the readily available porcinetrypsin, or alternatively bovine trypsin or a recombinant trypsin. Anyproteolytic method that provides substantially limited and selectivecleavage can be used, but preferred is an immobilized-trypsin column inorder to minimize contamination of the NGF preparation. The column isrun at a pH conducive to protease activity, preferably pH 5.5 to 8.5,more preferably 6.0 to 8.0, and most preferably 6.5 to 7.5.

In this example, glycosylated NGF was removed by HIC as discussedherein. Following an SP-Sepharose HP step as discussed herein in ExampleII but preferably using a 22 column volume 0.3 M to 0.55M salt gradient,a preferred composition of NGF for clinical use was obtained. Acomposition of greater than 70% 118 monomer, less than 10% 120 monomer,and less than 15% 117 monomer, as determined by RP-HPLC assays, can beobtained. Typically compositions that are greater than or equal to 90%118/118 rhNGF, more usually greater than or equal to 93% 118/118 rhNGFwith less than or equal to about 7% deamidated, isoAsp and oxidizedvariants are obtained. One means to achieve higher purity is to avoidselecting fractions with significant amounts of variants, such as may befound in the leading or trailing edges of the main neurotrophin peak,e.g. 118/118 rhNGF peak.

Example IV Partial Purification and Refolding of rbNT-4/5 from BacterialInclusion Bodies

In this example, starting with a 10 or 60 liter fermentation, rhNT-4/5was purified. The host used to produce recombinant human NT-4/5 in thefermentation described in this example was an E. coli strain designated27C7/pmNT5DT; although NT-4/5 produced from other strains and organismsis suitable for the purification process described herein. Theexpression plasmid used in this example contained the mature NT-4/5coding sequence under transcriptional and translational controlsequences required for expression of the NT-4/5 gene in E. coli. In theNT-4/5-expressing plasmid, the transcriptional sequences used forexpression of the gene in E. coli were provided by the alkalinephosphatase promoter sequence. The lambda to transcriptional terminatorwas situated adjacent to the NT-4/5 termination codon. Secretion of theprotein from the cytoplasm was directed by the STII signal sequence. Themajority of rhNT-4/5 was found in the cell periplasmic space asrefractile bodies. The plasmid conferred tetracycline resistance uponthe transformed host. The fermentation process was performed at 35°-39°C. and pH 7.0-7.8. The fermentation was allowed to proceed for 25-40hours, at which time the culture was chilled prior to harvest. Theculture was inactivated by heat treatment using a continuous-flowapparatus at 60° C. or using in-tank heat inactivation at thattemperature for 5-15 minutes. The heat-inactivated culture wascentrifuged using a AX Alpha-laval centrifuge or equivalent. The E. colicells were recovered in the pellet.

The E. coli cells, expressing recombinant human NT-4/5 in inclusionbodies, were lysed by standard means to prepare a paste containingNT-4/5 in inclusion bodies. No protease inhibitors were included in thebuffer.

To isolate the inclusion bodies from cell debris, the E. coli NT-5 pastewas resuspended in 0.02 M Tris, pH 8, 5 mM EDTA (10 ml of buffer/grampaste) using a rotary, mechanical dispersion device, for example aTurrax. The cell suspension was passed through a microfluidizer threetimes at 6000 psi. The resulting homogenate was centrifuged in a SorvallRC-3B centrifuge at 5000 rpm for about 45 min. Supernatant was discardedand the pellet was resuspended in 20 mM Tris, pH 8, 5 mM EDTA(Extraction buffer) using a turrax for 2 to 3 minutes at medium speed.The homogenate was centrifuged as described above. The pellet wasresuspended in extraction buffer and centrifuged as described above. Theresulting pellet(s) (referred to as NT-4/5 inclusion bodies orrefractile bodies) was stored at −70° C.

NT-4/5 was isolated from the inclusion bodies as follows. The inclusionbody pellets were suspended in 20 mM Tris, pH 8, 6M Urea, 25 mM DTT (10ml buffer/gram inclusion body) using a turrax at medium speed for about10 min. The suspension was stirred for 40 min at 2-8° C. and centrifugedin a Sorvall RC3B at 5000 rpm for about 45 min. PEI(poly-ethylene-imine) was added to 0.1% in the supernatant, which wasstirred at 2-8° C. for 30 minutes. The PEI precipitates nucleic acid andother acidic-charged molecules. The mixture was centrifuged in a SorvallRC3B at 5000 rpm for about 45 minutes. The PEI supernatant was loadedonto a DEFF Sepharose Fast Flow column (10 cm×14 cm; DEFF is a diethylaminoethyl resin) equilibrated in 0.02 M Tris, 6M Urea, 10 mM DTT, pH 8.An equivalent of 1 kg of solubilized refractile bodies was loaded ontothe DEFF column. Since reduced and denatured NT-4/5 does not bind to theDEFF resin, the flow through pool containing NT-4/5 and 6M urea, wascollected (FIG. 6) and the pH of the pool was lowered to 5.0 with aceticacid. The pH-adjusted DEFF flow through pool was loaded onto aS-Sepharose Fast Flow column (S refers to the SO3 functional group onthe resin) equilibrated in 20 mM acetate, pH 5, containing 6M urea,under which conditions NT-4/5 binds to the resin. After loading, theS-Sepharose Fast Flow column was washed with several column volumes ofequilibration buffer. The bound NT-4/5 was eluted with 0.5 M NaCl, 20 mMsodium acetate, 6M urea, pH 5 (FIG. 7). The 0.5 M NaCl SSFF pool wasdialyzed overnight against 20 mM Tris, 0.14 M NaCl, pH 8, conditionsthat allow NT-4/5 to refold albeit incorrectly. The misfolded rhNT-4/5molecules aggregated to form a precipitate.

The aggregated, misfolded rhNT-4/5 was processed to obtain correctlyfolded NT-4/5. The aggregated, misfolded rhNT-4/5 was collected bycentrifugation as a pellet. The pellet was resuspended in 0.2 M Tris, pH8, 4 M Urea, 5 mM DTT and stirred at 2-8° C. for about 1 to 2 hrs oruntil the pelt dissolved. The final protein concentration was adjustedto about 10 mg/ml protein based on the extinction coefficient 1.8 at 280nm. Oxidized glutathione was added to the solubilized pellet solution toa final concentration of 20 mM, followed by gentle stirring for 15 to 30min at 2-8° C. The oxidized glutathione reacts with the NT-4/5sulfhydryl groups to yield NT-4/5-S-glutathione mixed disulfide. TheNT-4/5-SG mixed disulfide were diluted to a final concentration of 0.1to 0.5 mg/ml protein in 100 mM Tris, 20 mM glycine, 15% PEG-300, 1MGuanidine HCI, pH 8.3. To initiate proper refolding of NT-4/5, cysteinewas added to the refold mixture at a concentration of 2 to 4 mM,followed by aeration (by bubbling through) of the solution with nitrogenor helium for 5 to 60 minutes before sealing the container to excludeoxygen. The refolding of NT-4/5 was allowed to proceed for 18 to 24 hrsat 2 to 8° C.

Alternatively, the rhNT-4/5 was refolded using sulfitolysis as follows.The inclusion body pellets (110 g) were suspended in 1.1 liter of 20 mMTris, 7M Urea, 10 mM glycine, 100 mM sodium sulfite, 10 mM sodiumtetrathionate, and solubilized using a turrax for 10 minutes at mediumspeed. The mixture (1260 mL) was then stirred at 2 to 8° C. for 45minutes. PEI was added to a final concentration of 0.1% PEI. The mixturewas stirred for an additional 30 minutes at 4° C. and centrifuged for 45min at 5500 rpm in a RC3B centrifuge. The supernatant was loaded onto aDEFF column (4.4 cm×25 cm) equilibrated in 20 mM Tris, 6M Urea, pH 8.The DEFF flow through was adjusted to pH 5 with acetic acid and loadedonto a S-Sepharose Fast Flow column (4.4 cm×25 cm) equilibrated with 20mM acetate, 6 M urea, pH 5. The NT-4/5 was eluted with 25 mM MOPSO, 0.5M NaCl, pH 7.

The SSFF 0.5 M sodium chloride pool containing sulfonylated rhNT-4/5 wasdiluted to about 0.1 mg/ml protein and adjusted to 1M guanidinehydrochloride, 100 mM Tris, 20 mM glycine, 15% PEG-300, pH 8.3. Therefolding of NT-4/5 was started by the addition of 2 to 4 mM cysteine.The refolding reaction was essentially complete within 24 hrs. Aerationwith an inert gas, e.g. helium or nitrogen, to replace oxygen from thesolution, can be optionally performed.

Example V Isolation of Correctly Folded rhNT-4/5 From Conformational(Misfolded) Variants

The refold mixture of rhNT-4/5 of Example IV was dialyzed against a pH 4to 5 solution overnight to remove guanidine and other reagents. Toclarify the solution, the solution was either centrifuged for 45 minutesat 5000 rpm or passed through a 0.2 um filter.

The clarified supernatant, containing 0.5 to 5 grams of protein, wasadjusted to pH 3 to 5 by addition of glacial acetic acid and was eitherloaded onto a C4 RP-HPLC column or stored frozen at −20° C. until readyfor purification. In this example, the acidified and clarified solutionwas loaded onto a C4 RP-HPLC (3 cm×50 cm) column, to which resin thefolded rhNT-4/5 bound. The correctly folded NT-4/5 was eluted using anacetonitrile gradient in a 0.05 Trifluoroacetic acid (TFA) solventsystem: a 26 to 40% acetonitrile gradient (over a 95 minute period) in0.05% TFA at a flow rate of 25 ml/min. Fractions were collected at 1 to1.5 minute intervals (FIG. 8). Fractions were analyzed for correctlyfolded NT-4/5 by comparing elution time on an analytical C4 HPLC Vydac(0.21×15 cm) column to that of a correctly folded NT-4/5 standard (FIG.9). Standard correctly folded, intact NT-4/5 typically eluted at 19minutes at a flow rate of 2.5 ml per minute with a 0.5% TFA/acetonitrilebuffer system. The fractions containing correctly folded rhNT-4/5 werepooled and pH-adjusted to pH 5 to 7. This pool of correctly foldedrhNT-4/5 also contained carbamylated and N-terminal clipped forms ofNT-4/5.

The preparative reverse-phase liquid chromatography resin is preferablya medium having a particle diameter of about 10-40 microns, a pore sizeof about 200-400 Angstroms, and a C4, C8, or C 18 alkyl group. Morepreferably, the resin has a particle diameter of about 15-40 micron anda pore size of about 300 Angstroms, and is a C4 silica medium.

Example VI An Alternative Isolation of Correctly Folded rhNT-4/5 FromConformational (Misfolded) Variants

The refolded rhNT-4/5 mixture of Example IV was concentratedapproximately 10-fold using a Millipore-Pellicon ultrafiltration systemwith 20 square foot cellulose (or polysulfone or equivalent) membranewith a 10 kD-molecular weight cut-off. The concentrated mixture waseither dialyzed overnight against 50 L of 50 mM acetate, pH 5.5, 50 mMNaCl or diafiltered into 50 mM acetate, 50 mM NaCl, pH 5.5, prior tofiltering through a 0.2 micron membrane.

The filtered refolding mixture was adjusted to 2.5 M NaCl, 20 mM MOPSO,pH 7 and loaded onto a HIC column, phenyl Toyopearl 650M column (10cm×19 cm), previously equilibrated in 2.5 M NaCl, 20 mM MOPSO, pH 7. Thecolumn was then washed with equilibration buffer. Some misfolded formsof the rhNT-4/5 molecule eluted in the flow-through fractions, whileother misfolded forms were eluted at high concentrations of organicsolvents such as 20 to 40% reagent alcohol. The correctly foldedrhNT-4/5 was eluted from the phenyl column using 2 M sodium chloride,10% reagent alcohol, pH 7 (FIG. 10). Other phenyl resins such as phenylSepharose can be used in place of the Toyopearl backbone. Saltsdiscussed herein, including ammonium sulfate, citrate, acetate, andpotassium chloride can be used. Depending on the salt used, the saltconcentration is typically 1 M to 3 M, with 2.5 M NaCl being preferredfor loading and 2M NaCl preferred for elution when organic solvent ispresent. Preferably, a lowering of the salt concentration is used toelute and separate a neurotrophin and its variants. In order to achieveelution, the salt concentration in the elution buffer is typically lowerthan that in the loading buffer, but it can be the same concentrationwhen compensated for with organic solvent. In addition, the use oforganic solvent has another advantage, as has been found herein, thatthe addition of an organic solvent improves the elution pattern byresulting in narrower peak profiles. In addition to ethanol, otherorganic solvents discussed herein can be used, including propanol,isopropanol, and lower alkylene glycols, such as propylene glycol,ethylene glycol and hexylene glycol. The organic solvent at 5 to 25%(v/v), more preferably 5 to 20% (v/v), even more preferably 5 to 15%,will typically elute a correctly folded neurotrophin. The elution withorganic solvent can be either gradient or step-wise. The pH range ispreferably near neutral to slightly acidic, from pH 5 to 8, morepreferably pH 5.5 to 7.5, and most preferably pH 7. Any of the buffersdiscussed herein, including MOPSO, MOPS, HEPES, phosphate, citrate,ammonium, acetate, can be used as long as they buffer at the desired pH.

Example VII Purification of Correctly Folded rhNT-4/5 From ChemicalVariants

Separation of correctly folded, intact rhNT-4/5 from its chemicalvariants, including carbamylated and N-terminal clipped forms ofrhNT-4/5, was accomplished by high performance cation-exchangechromatography using SP-Sepharose HP resin or PolyCat a HPLC resin.

When the C4 RP-HPLC column was used to remove misfolded variants, the C4HPLC pool was adjusted to pH 5 to 7 and loaded onto a 7 cm×19 cmSP-sepharose HP column equilibrated in 20 mM succinate, pH 6, 5% reagentalcohol, 0.2 M NaCl. The resin with bound NT-4/5 was washed withequilibration buffer. The bound rbNT-4/5 was eluted and separated fromthe carbamylated and N-terminal clipped forms using a 22 column volume(CV) gradient from 0.2 M NaCl to 0.4 M NaCl at pH 6 (i.e., salt gradientin equilibration buffer) (FIG. 11). Fractions containing NT-4/5 werepooled and formulated into 0.05 M acetate, pH 4 to 5. Intact rhNT-4/5was identified and distinguished from the variants in the fractionspreferably by analytical RP-HPLC, or by SDS-PAGE, as discussed herein,compared against standard.

Alternatively, the variant forms of NT-4/5 were removed by highperformance cation-exchange HPLC on a polyaspartic acid column (PolyCATa, PolyLC, Columbia, Md.) (9.4×200 mm) (FIG. 12). The C4 HPLC pool wasadjusted to pH 5 to 6 and then loaded onto the Polycat a column. Thechromatography conditions were: Buffer A was 20 mM phosphate, 5%acetonitrile, pH 6; Buffer B was 20 mM phosphate, 5% acetonitrile, 0.8 MKCl, pH 6. The rhNT-4/5 was eluted using a gradient of 25 to 60% BufferB over 65 minutes (FIG. 12). Fractions were collected at 1 minuteintervals and analyzed by analytical C4 IIPLC as described above.

When HIC was used to remove misfolded variants (Example VI above), thecorrectly folded NT-4/5 pool was dialyzed overnight into 20 mMsuccinate, 0.1 M NaCl, 5% reagent alcohol, pH 6 orultrafiltered/diafiltered into the 20 mM succinate buffer. The dialyzedor UF/DF pool was then loaded onto a SP-Sepharose HP or PolyCAT a columnas described above.

Formulation

Fractions containing correctly folded, intact NT-4/5 (from theSP-Sepharose HP or PolyCAT a HPLC step) were pooled and concentrated to1 to 5 mg/ml in 20 mM acetate, pH 4 to 5 formulation buffer.Alternatively, the NT-4/5 was formulated usingultrafiltration/diafiltration.

The final bulk solution was analyzed by amino acid analysis, N-terminalsequence analysis, mass spectrometry, SDS-PAGE (FIG. 13) and biologicalassays, a kinase receptor activation (KIRA) assay, which detects NT-4/5activation of autophosphorylation of its tyrosine kinase receptor (trkB)located in a cell membrane, was used to characterize the purifiedrhNT-4/5. CHO cells expressing trkB with a gD tag were used. WO95/14930, published Jun. 1, 1995, describes the KIRA assay and isincorporated herein by reference. The rhNT-4/5 had an EC50 of 12.6 ng/mlin this assay. Typically the EC50 of correctly, folded, intact NT-4/5,purified as described herein, is 5 to 30, more preferably 10 to 20.

Purity of rhNT-4/5 with respect to non-NT-4/5 proteins was typically 90to 99 percent. Homogeneity of NT-4/5 with respect to carbamylated andN-terminal clipped variants was from 90 to 99 percent. Most typically,and preferably, the purity and homogeneity are 99% or greater.

Example VIII Initial Purification, Refolding and Final Purification ofrhNT-3 from Bacterial Inclusion Bodies

To isolate the inclusion bodies from cell debris, the E. coli NT-3 paste(1 kg) was resuspended in 10 L of 100 mM sodium acetate, pH 5 using arotary, mechanical dispersion device, for example a turrax. The cellsuspension was passed through a microfluidizer three times at 6000 psi.The resulting homogenate was centrifuged in a Sorvall RC-3B centrifugeat 5000 rpm for 30 minutes.

NT-3 was isolated from the inclusion bodies as follows. The inclusionbody pellets were suspended 100 mM Tris, 100 mM NaCl, 5 mM EDTA, 100 mMsodium sulfite, 10 mM sodium tetrathionate, 7.5 M urea, pH 8.3 (10ml/gram of inclusion body) using a turrax at medium speed for about 10min. The suspension was stirred for about one hour at 2-8° C. PEI(polyethyleneimine) was added to approximately 0.15% (finalconcentration) and stirred at 2-8° C. for 30 minutes. The mixture wascentrifuged in a Sorval RC3B at 5000 rpm for abut 30 minutes. Thesupernatant was filtered with a Gelnan Preflow cartridge. The filteredsupernatant was diluted with 3 volumes of S-Sepharose Fast Flowequilibration buffer (50 mM sodium acetate, 5 M urea, pH 5). The dilutedfiltered supernatant (conductivity less than 7 mS) is loaded onto anS-Sepharose FF column equilibrated with 50 mM sodium acetate, 5 M urea,pH 5.0. The column was first washed with 50 mM sodium acetate, 5M Urea,pH 5, followed by 50 mM MOPS, 5 M urea, 10 mM glycine, pH 7.0.Sulfitolyzed NT-3 was eluted from the column using a 10 column volumegradient from 0-0.6 M NaCl in 50 mM in 50 mM MOPS, 5 M urea, 10 mMglycine, pH 7.

Partially purified NT-3 was refolded by diluting the S-Sepharose FF poolto approximately 0.1 mg/ml protein in refolding buffer containing 0.1 MTris, 2 M urea, 0.1 M NaCl, 15% PEG 300, 10 mM glycine, 25 mMethanolamine, pH 9.1. Refolding was initiated by adding cysteine toapproximately 5 mM and stirring for 2-5 days at 2-8° C. Optionally, therefolding buffer can be sparged with lie or Argon to reduce the oxygenconcentration in the refolding solution.

The pH of the refolded pool was adjusted to pH 7, filtered and loadedonto a Macroprep High S cation-exchange chromatography columnequilibrated in 50 mM HEPES, pH 7. After loading the pH adjusted refoldpool to the Macroprep column, the column was first washed with 50 mMMOPS, pH 7 followed by 50 mM MOPS, 0.1 M TMAC, 0.3 M NaCl, pH 7. NT-3was eluted with 50 mM MOPS, 0.25 M TMAC, 1.5 M NaCl, pH 7.

The Macroprep pool was then further purified on Phenyl Sepharose FastFlow High Substitution column. The Phenyl column was equilibrated in 50mM HEPES, 1.5 M NaCl, pH 7 and the macroprep pool was directly loaded tothe Phenyl column. The column was washed with equilibration buffer andthen the correctly refolded NT-3 was eluted using a 15 column volumegradient going from 50 mM HEPES, 1.5 M NaCl, pH 7 to 50 mM HEPES, 10%reagent alcohol, pH 7. Fractions were analyzed by either C4 HPLC or bySDS-PAGE and the fractions containing correctly-folded NT-3 were pooled.

The Phenyl pool was diluted to less than 25 mS (typically about 2volumes with water) and loaded onto a SP-Sepharose HP column previouslyequilibrated in 25 mM MOPSO, pH 7. The column was first washed withequilibration buffer and NT-3 was eluted from the column using a 20column volume gradient going from 0.35 M TMAC to 0.65 M TMAC in 25 mMMOPSO, pH 7. Fractions containing rhNT-3 (as judged by C4 HPLC assay)were pooled.

The SP-Sepharose HP pool was concentrated to about 1 mg/ml on a 10,000molecular weight membrane and then diafiltered with 6 volume of 10 mMacetate, 140 mM NaCl, pH 5.0.

6 1 242 PRT Homo sapien 1 Pro Met Ser Met Leu Phe Tyr Thr Leu Ile ThrAla Phe Leu Ile Gly 1 5 10 15 Ile Gln Ala Glu Pro His Ser Glu Ser AsnVal Pro Ala Gly His Thr 20 25 30 Ile Pro Gln Val His Trp Thr Lys Leu GlnHis Ser Leu Asp Thr Ala 35 40 45 Leu Arg Arg Ala Arg Ser Ala Pro Ala AlaAla Ile Ala Ala Arg Val 50 55 60 Ala Gly Gln Thr Arg Asn Ile Thr Val AspPro Arg Leu Phe Lys Lys 65 70 75 80 Arg Arg Leu Arg Ser Pro Arg Val LeuPhe Ser Thr Gln Pro Pro Arg 85 90 95 Glu Ala Ala Asp Thr Gln Asp Leu AspPhe Glu Val Gly Gly Ala Ala 100 105 110 Pro Phe Asn Arg Thr His Arg SerLys Arg Ser Ser Ser His Pro Ile 115 120 125 Phe His Arg Gly Glu Phe SerVal Cys Asp Ser Val Ser Val Trp Val 130 135 140 Gly Asp Lys Thr Thr AlaThr Asp Ile Lys Gly Lys Glu Val Met Val 145 150 155 160 Leu Gly Glu ValAsn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe 165 170 175 Glu Thr LysCys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly 180 185 190 Ile AspSer Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe 195 200 205 ValLys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile 210 215 220Arg Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg 225 230235 240 Arg Ala 2 121 PRT Homo sapien 2 Pro Ser Ser Ser His Pro Ile PheHis Arg Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Val Ser Val Trp ValGly Asp Lys Thr Thr Ala Thr Asp Ile 20 25 30 Lys Gly Lys Glu Val Met ValLeu Gly Glu Val Asn Ile Asn Asn Ser 35 40 45 Val Phe Arg Gln Tyr Phe PheGlu Thr Lys Cys Arg Asp Pro Asn Pro 50 55 60 Val Asp Ser Gly Cys Arg GlyIle Asp Ser Lys His Trp Asn Ser Tyr 65 70 75 80 Cys Thr Thr Thr His ThrPhe Val Lys Ala Leu Thr Met Asp Gly Lys 85 90 95 Gln Ala Ala Trp Arg PheIle Arg Ile Asp Thr Ala Cys Val Cys Val 100 105 110 Leu Ser Arg Lys AlaVal Arg Arg Ala 115 120 3 121 PRT mouse 3 Pro Ser Ser Thr His Pro ValPhe His Met Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Val Ser Val TrpVal Gly Asp Lys Thr Thr Ala Thr Asp Ile 20 25 30 Lys Gly Lys Glu Val ThrVal Leu Ala Glu Val Asn Ile Asn Asn Ser 35 40 45 Val Phe Arg Gln Tyr PhePhe Glu Thr Lys Cys Arg Ala Ser Asn Pro 50 55 60 Val Glu Ser Gly Cys ArgGly Ile Asp Ser Lys His Trp Asn Ser Tyr 65 70 75 80 Cys Thr Thr Thr HisThr Phe Val Lys Ala Leu Thr Thr Asp Glu Lys 85 90 95 Gln Ala Ala Trp ArgPhe Ile Arg Ile Asp Thr Ala Cys Val Cys Val 100 105 110 Leu Ser Arg LysAla Thr Arg Arg Gly 115 120 4 119 PRT Homo sapien 4 Pro His Ser Asp ProAla Arg Arg Gly Glu Leu Ser Val Cys Asp Ser 1 5 10 15 Ile Ser Glu TrpVal Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met 20 25 30 Ser Gly Gly ThrVal Thr Val Leu Glu Lys Val Pro Val Ser Lys Gly 35 40 45 Gln Leu Lys GlnTyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr 50 55 60 Thr Lys Glu GlyCys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln 65 70 75 80 Cys Arg ThrThr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys 85 90 95 Lys Arg IleGly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Thr 100 105 110 Leu ThrIle Lys Arg Gly Arg 115 5 120 PRT Homo sapien 5 Pro Tyr Ala Glu His LysSer His Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser Glu Ser Leu TrpVal Thr Asp Lys Ser Ser Ala Ile Asp Ile Arg 20 25 30 Gly His Gln Val ThrVal Leu Gly Glu Ile Lys Thr Gly Asn Ser Pro 35 40 45 Val Lys Gln Tyr PheTyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val 50 55 60 Lys Asn Gly Cys ArgGly Ile Asp Asp Lys His Trp Asn Ser Gln Cys 65 70 75 80 Lys Thr Ser GlnThr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys 85 90 95 Leu Val Gly TrpArg Trp Ile Arg Ile Asp Thr Ser Cys Val Ser Ala 100 105 110 Leu Ser ArgLys Ile Gly Arg Thr 115 120 6 130 PRT Homo sapien 6 Gly Val Ser Glu ThrAla Pro Ala Ser Arg Arg Gly Glu Leu Ala Val 1 5 10 15 Cys Asp Ala ValSer Gly Trp Val Thr Asp Arg Arg Thr Ala Val Asp 20 25 30 Leu Arg Gly ArgGlu Val Glu Val Leu Gly Glu Val Pro Ala Ala Gly 35 40 45 Gly Ser Pro LeuArg Gln Tyr Phe Phe Glu Thr Arg Cys Lys Ala Asp 50 55 60 Asn Ala Glu GluGly Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly 65 70 75 80 Val Asp ArgArg His Trp Val Ser Glu Cys Lys Ala Lys Gln Ser Tyr 85 90 95 Val Arg AlaLeu Thr Ala His Ala Gln Gly Arg Val Gly Trp Arg Trp 100 105 110 Ile ArgIle Asp Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly 115 120 125 ArgAla 130

What is claimed is:
 1. A process to isolate a neurotrophin from amixture containing variants of said neurotrophin, wherein the processcomprises: a) purifying a neurotrophin mixture; b) loading the mixturecontaining the neurotrophin onto a hydrophobic interactionchromatography resin; c) eluting the neurotrophin from the resin with anelution buffer under conditions in which the neurotrophin separates fromthe variant; and d) collecting the neurotrophin.
 2. The process of claim1, wherein said purifying comprises affinity chromatography.
 3. Theprocess of claim 1, wherein said purifying comprises purifying withchromatography on silica.
 4. The process of claim 1, wherein saidpurifying comprises purifying with chromatography on heparin agarose. 5.The process of claim 1, wherein said purifying comprises purifying withchromatography on an anion exchange resin.
 6. The process of claim 1,wherein said purifying comprises purifying with chromatofocusing.
 7. Theprocess of claim 1, wherein said purification comprises purifying withpreparative sodium deodecylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE).
 8. A composition prepared by the method of claim 1comprising a neurotrophin.
 9. The process of claim 1, wherein saidloading of said mixture comprises loading a mixture having a volume ofat least about 700 mL onto a hydrophobic interaction chromatographyresin.
 10. The process of claim 1, wherein said loading of said mixturecomprises loading a mixture having a volume of at least about 1200 mLonto a hydrophobic interaction chromatography resin.
 11. The process ofclaim 1, wherein said purifying comprises purifying with chromatographyon a cation exchange resin.
 12. The process of claim 11, wherein saidcation exchange resin comprises a polyaspartic acid column.
 13. Acomposition prepared by the method of claim 1 comprising a mixture ofneurotrophins.
 14. The composition of claim 13 wherein said mixture ofneurotrophins comprises nerve growth factor (NGF) and at least one otherneurotrophin.
 15. The composition of claim 13 wherein said mixture ofneurotrophins comprises at least two neurotrophins selected from thegroup consisting of nerve growth factor (NGF), neurotrophin-4/5(NT-4/5), neurotrophin-3 (NT-3), brain-derived neurotrophic factor(BDNr) and homologs thereof.
 16. The process of claim 1, wherein theresin comprises a phenyl functional group.
 17. The process of claim 16,wherein the resin is a sulphopropyl agarose high performance, polyaspartic acid resin, polysulfoethyl cation exchange resin, orsulfoisobutyl (SO₃) resin.
 18. The process of claim 16, furthercomprising the step of separating the neurotrophin from a misfoldedvariant of that neurotrophin using preparative reversed-phase liquidchromatography resin.
 19. The process of claim 18, wherein the resincontains a carbon at position 4 (C4) functional group.
 20. A process toisolate a neurotrophin from a mixture containing variants of saidneurotrophin, wherein the process comprises: a) purifying a neurotrophinmixture prepared from cells; b) loading the mixture containing theneurotrophin onto a hydrophobic interaction chromatography resin; and c)eluting the neurotrophin from the resin with an elution buffer underconditions in which the neurotrophin separates from the variant.